A-kinase anchoring proteins AKAP1, -4, -10 and

Transcription

A-kinase anchoring proteins AKAP1, -4, -10
and -11 with different subcellular localizations in
Sertoli cells and their roles in male fertility
Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen
by
M. Sc. Wang, Wenwen
of
Qingdao, China
Gießen (2016)
From the Institute for Anatomy and Cell Biology,
Medical Cell Biology
Director: Prof. Dr. med. Eveline Baumgart-Vogt
of the Faculty of Medicine of the Justus Liebig University Giessen
First Supervisor: Prof. Dr. med. Eveline Baumgart-Vogt
Second Supervisor: Prof. Dr. habil. Christiane Herden
Committee Members: Prof. Dr. Klaus-Dieter Schlüter, Prof. Dr. med. Eveline
Baumgart-Vogt, Prof. Dr. habil. Christiane Herden
Date of Doctoral Defense: 18th May 2016
II
Declaration
“I declare that I have completed this dissertation single-handedly without the
unauthorized help of a second party and only with the assistance acknowledged therein.
I have appropriately acknowledged and referenced all text passages that are derived
literally from or are based on the content of published or unpublished work of others,
and all information that relates to verbal communications. I have abided by the
principles of good scientific conduct laid down in the charter of the Justus Liebig
University of Giessen in carrying out the investigations described in the dissertation.”
Wenwen Wang
Date: 18th May 2016
Place: Gießen, Germany
III
Contents
1
INTRODUCTION ............................................................................................................................ 7
1.1
THE WORLDWIDE SOCIAL PROBLEM OF INFERTILITY.............................................................................. 7
1.1.1
1.2
Morphology and function of Sertoli cells ............................................................................. 8
1.2.2
Models to study Sertoli cells in vitro ................................................................................... 10
1.2.3
Sertoli cell function regulation ............................................................................................. 10
AKAPS: SCAFFOLDS FOR LOCAL SIGNALING.......................................................................................... 13
1.3.1
AKAP binding features and signaling cross-talk ........................................................... 14
1.3.2
Structure of AKAP-PKA interaction .................................................................................... 15
1.3.3
Diversity and evolution within the AKAP family ........................................................... 17
1.4
A CLOSER LOOK TO FOUR AKAPS USED IN THIS STUDY ....................................................................... 18
1.4.1
AKAP11 (=AKAP220)................................................................................................................ 18
1.4.2
AKAP1 ............................................................................................................................................. 21
1.4.3
AKAP4 ............................................................................................................................................. 28
1.4.4
AKAP10 .......................................................................................................................................... 29
1.5
THE FUNCTION OF AKAP1, AKAP4, AKAP10 AND AKAP11 IN GENITAL ORGANS ................... 30
1.5.1
Steroid hormone biosynthesis ............................................................................................... 30
1.5.2
Sperm development .................................................................................................................. 31
1.5.3
Oocyte maturation .................................................................................................................... 31
1.5.4
Prostate cancer ........................................................................................................................... 32
1.6
PEROXISOMES AND INFERTILITY ............................................................................................................. 32
1.6.1
Metabolic pathways in peroxisomes involved in male fertility ................................ 32
1.6.2
Peroxisomal matrix protein translocation and import machinery ........................ 34
1.7
3
SERTOLI CELLS............................................................................................................................................... 8
1.2.1
1.3
2
Cause of male infertility ............................................................................................................. 7
AIMS OF THE THESIS .................................................................................................................................. 35
MATERIALS .................................................................................................................................. 36
2.1
CELL LINE AND MEDIUM............................................................................................................................ 36
2.2
ROUTINE MATERIALS................................................................................................................................ 36
2.3
BUFFER RECIPES......................................................................................................................................... 39
2.4
AKAP PLASMIDS ........................................................................................................................................ 40
2.5
PRIMERS ...................................................................................................................................................... 42
2.6
ANTIBODY LIST ........................................................................................................................................... 47
METHODS ..................................................................................................................................... 49
3.1
BIOINFOMATIC SCREENING FOR THE AKAPS WITH PUTATIVE PTS1 SIGNALS ............................... 49
3.1.1
Scan of AKAP sequences for the consensus PTS1 motif .............................................. 49
3.1.2
Use of ClustalX 2.0 & MEGA 4.0.2 programs to create an evolutionary tree ...... 49
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3.1.3
Use of Python 2.7.5 & R i386 3.0.1 programs to test AKAP1, AKAP4, AKAP10
through PTS1 predictor .............................................................................................................................................. 50
3.2
3.2.1
Cell culture routine ................................................................................................................... 52
3.2.2
Cell number and viability examination ............................................................................. 52
3.2.3
Isolation of total RNA from TM4 cells ................................................................................ 53
3.3
RT-PCR ...................................................................................................................................................... 53
3.4
SDS-PAGE AND WESTERN BLOTS ......................................................................................................... 54
3.5
PEROXISOME PURIFICATION BY OPTIPREP™ DENSITY GRADIENT CENTRIFUGATION..................... 54
3.6
RAT DISSECTION AND RNA ISOLATION .................................................................................................. 59
3.6.1
Anaesthesia for rats and dissection of different organs ............................................. 59
3.6.2
Tissue total RNA isolation ...................................................................................................... 59
3.6.3
Ethanol precipitation to concentrate total RNA ........................................................... 60
3.7
WORKFLOW FOR NORTHERN BLOTTING................................................................................................ 60
3.7.1
Plasmid construction and probe generation by in vitro RNA transcription ...... 62
3.7.2
Dot blot assay for detection of DIG-labeling efficiency ............................................... 62
3.7.3
RNA samples and agarose gel preparation ..................................................................... 63
3.7.4
Protocol of Northern blotting ............................................................................................... 63
3.8
4
CELL CULTURE ............................................................................................................................................ 52
CONSTRUCTION OF MYC-TAGGED AKAP PLASMIDS AND EXPRESSION IN TM4 CELLS................... 64
3.8.1
Construction of myc-tagged AKAPs and GFP plasmids ............................................... 65
3.8.2
Procedures for bacterial transformation ......................................................................... 68
3.8.3
Point mutation assay ................................................................................................................ 70
3.8.4
Coating cover slips with poly-L-lysine ............................................................................... 71
3.8.5
Transient transfection of TM4 cells .................................................................................... 72
3.8.6
Immunofluorescence protocol .............................................................................................. 72
RESULTS ........................................................................................................................................ 73
4.1
AKAP FAMILY MEMBERS WITH PUTATIVE PTS1 SIGNALS ................................................................. 73
4.1.1
AKAP sequence retrieval and PTS1 search matrix ....................................................... 73
4.1.2
Phylogenetic analysis of AKAP proteins containing a putative PTS1 signal ...... 74
4.2
TM4 SERTOLI CELL CULTURE STIMULATION WITH FSH TREATMENT ............................................. 76
4.2.1
Cell culture condition optimization .................................................................................... 76
4.2.2
Effect of FSH stimulation on the peroxisome gene expression ................................. 77
4.3
ISOLATION OF PEROXISOMES FROM TM4 CELLS................................................................................... 81
4.4
ANALYSIS OF AKAKP11 (=AKAP220) CDNA WITH GENOMIC SEQUENCES ................................ 84
4.5
NORTHERN BLOTTING ............................................................................................................................... 87
4.5.1
Total RNA preparation for Northern blotting ................................................................ 87
4.5.2
Specific RNA probe generation and validation .............................................................. 88
4.5.3
Rat tissue total RNA Northern blot test ............................................................................ 95
4.5.4
Rat total RNA RT-PCR .............................................................................................................. 96
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4.5.5
4.6
5
Concentrated total RNA and PCR-generated probes ................................................... 97
BASAL ANALYSIS AND OVER EXPRESSION OF AKAP1, AKAP4, AKAP10 IN TM4 CELLS ........... 98
4.6.1
Basic analysis of AKAP1, AKAP4, AKAP10 ....................................................................... 98
4.6.2
Cloning and immunolocalization of myc-tagged AKAPs .......................................... 106
DISCUSSION ............................................................................................................................... 121
5.1
AKAP220 (-CRL) IS PROBABLY AN ARTIFACT................................................................................. 121
5.2
PTS1 PREDICTION IS USEFUL FOR SEARCHING NOVEL PEROXISOMAL PROTEINS ......................... 122
5.3
EXCEPTIONS TO THE RULES OF PTS1 PREDICTION: ALTERNATIVE TARGETING OF PEROXISOMAL
MATRIX PROTEINS ................................................................................................................................................................ 125
5.4
AKAP1 AND MITOCHONDRIAL FRAGMENTATION ............................................................................. 126
5.5
MITOCHONDRIAL FUSION AND FISSION MECHANISMS....................................................................... 127
5.6
CONNECTION BETWEEN PEROXISOMES AND MITOCHONDRIA ......................................................... 128
5.7
THE ROLE OF AKAP1-STAR INTERACTION IN SERTOLI CELLS ...................................................... 129
5.8
AKAP4 DETECTION AND IN VITRO LOCALIZATION ............................................................................ 131
6
OUTLOOK (FUTURE PLAN) .................................................................................................. 132
7
SUMMARY .................................................................................................................................. 133
8
ZUSAMMENFASSUNG ............................................................................................................. 136
9
LIST OF ABBREVIATIONS ..................................................................................................... 139
10
FIGURE INDEX .......................................................................................................................... 143
11
TABEL INDEX ............................................................................................................................ 147
12
SUPPLEMENTAL DATA .......................................................................................................... 148
13
ACKNOWLEDGEMENTS ......................................................................................................... 159
14
CURRICULUM VITAE .............................................................................................................. 161
15
REFERENCE ............................................................................................................................... 162
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1 Introduction
1.1 The worldwide social problem of infertility
According to the world health organization (WHO), a couple can be considered
infertile if it is not able to conceive after having unprotected intercourse continuously
for more than one year (Tvrda et al., 2015). Infertility has remained a global concern
since the first demographic survey provided by the WHO in 2002. On the average, 812% of sexually matured couples suffer from childlessness all over the world. In several
regions, including Africa, South and Central Asia, Central and Eastern Europe, the
infertility rates reach as high as 30%. Correspondingly, a global growth of in vitro
fertilization (IVF) services and assisted reproductive techniques (ARTs) are being
recorded (Inhorn and Patrizio, 2015). Therefore a better understanding of the processes
that lead to female and male infertility became of crucial importance. Although in more
than half of these cases, the reason for the childlessness is due to the male partner,
research on male infertility remains a neglected field. One of the most important
university centers, where laboratory research is currently conducted to elucidate the
molecular and metabolic causes that can lead to infertility in men, is the Hessian
Reproduction Center in Giessen, Germany.
1.1.1 Cause of male infertility
Male infertility can result from genetic defect such as chromosome disorder or
epigenetic factors e.g. incorrect gene transcription induced by DNA methylation and
histone modification. Abnormal expression of genes on Y chromosome may cause
oligozoospermia or azoospermia (Reijo et al., 1996). Moreover, male patients with
Klinefelter syndrome (47, XXY) suffer from hypogonadism and Down syndrome (47,
XX/XY, +21) patients often show poor testicular development (Hafeez et al., 2007). An
example for epigenetic alterations has been found in the NANOG promoter, a germ cell
tumor marker. This promoter has been observed to be hypomethylated in spermatogonia
and hypermethylated in spermatozoa, and its methylation state corresponds to the
differentiation of testicular tumor cells (Nettersheim et al., 2011). Additionally, many
environmental factors have been suspected to cause male infertility such as chemical
and physical agents from daily life activities, including heavy metals, pesticides such as
cypermethrin (Jin et al., 2011), organochlorides, radiation and plastics like bisphenol A
Page | 7
and plasticizers (Marques-Pinto and Carvalho, 2013). Certain chemicals with hormonal
properties commonly referred to as endocrine disrupting agents (EDAs) bind membrane
receptors preventing the endogenous steroid hormone synthesis, secretion and
metabolism. Sertoli cells known as the testicular nurse cells and key players during
spermatogenesis, have been found to be severely disrupted by EDAs accumulation.
Furthermore, exposure to EDAs leads to inhibition of follicle-stimulating hormone
(FSH) synthesis, which causes a decline in Sertoli cell number. The Sertoli cell
malfunction in turn leads to disruption of the spermatogenesis and probably induces
Testicular Dysgenesis Syndrome (TDS) (Tvrda et al., 2015).
1.2 Sertoli cells
1.2.1 Morphology and function of Sertoli cells
Sertoli cells are located in the basement membrane of seminiferous tubules in
the testis (Figure 1) and play a crucial role in spermatogenesis, male phenotypic
development, and the hypothalamic-pituitary-gonadal axis (Sharpe et al., 2003; Skinner
and Griswold, 2004). These cells are closely connected to each other via tight junctions,
forming the blood-testis barrier (BTB) also termed Sertoli cell barrier (SCB). SCB
controls the chemical composition of the luminal fluid and prevents cytotoxic agents
from penetrating the seminiferous tubules. Sertoli cells directly interact with male
germ cells and support their development by structural protection and nutrition.
Therefore, Sertoli cells are considered as support- or nurse cells (Griswold et al., 1987;
Guo et al., 2015; Nenicu, 2010; Nenicu et al., 2007).
Sertoli cells also play a key role in secretion of regulatory factors, e.g. growth
factors, hormones and cytokines, and the FSH response (Guo et al., 2015; McGuinness
et al., 1994). Besides the support to spermatogenesis, the presence of Sertoli cells is also
required to maintain the normal level of germ cell and Leydig cell population as well as
functional peritubular myoid cells in the adult male testis (Rebourcet et al., 2014). The
total number of Sertoli cells will determine the efficiency of sperm production and
testicular size. There is a positive correlation between the number of Sertoli cells and
testis weight, the percentage and volume of blood vessels, the seminiferous epithelium
volume and the daily number of produced germ cells (Leal et al., 2004). For these
reasons any disturbance of these cells could potentially cause infertility or an androgenrelated endocrine disorder.
Page | 8
Figure 1 Schematic illustration of testis structure (Figure 1A) and the hypothalamic-pituitary-testicular
axis (Figure 1B). Figure 1A is taken from website of the “School of Biological Sciences at University of
Texas at Austin” http://www.zo.utexas.edu/faculty/sjasper/images/f36.12.jpg and Figure 1B is taken
from the website https://imueos.wordpress.com/2010/05/24/spermatogenesis-x/.
Page | 9
1.2.2 Models to study Sertoli cells in vitro
For in vitro studies, primary Sertoli cells are often used. These cells are usually
isolated from the immature testis, and can be obtained with a relatively high purity
(Nenicu et al., 2007). However, compared to aged cells, these cells have significant
differences in the cell structure, transferrin expression level, and sensitivity to
androgens (Anway et al., 2003). Although a method of the isolation of Sertoli cells from
adult testis was recently established, these cells are often contaminated with germ cells
or peritubular cells. Moreover, these primary Sertoli cells start to lose the
responsiveness to hormones such as FSH within 6-10 days in culture (McGuinness et
al., 1994). Therefore, in several studies the TM4 cell line was used, which provides a
good model system to study Sertoli cell functions (Skinner and Griswold, 2004).
The TM4 cell line was established from immature mouse Sertoli cell cultures,
which was successfully used as a feeder layer for isolated original germ cells. Compared
with primary Sertoli cells, the TM4 cells are less sensitive to FSH stimulation.
Additionally, TM4 cells can inhibit testosterone production in Leydig cells, which is
rare in vivo. Nevertheless, TM4 cells still possess the major characteristics of primary
immature Sertoli cells, thereby providing a good tool to study Sertoli cell line signal
transduction processes (Skinner and Griswold, 2004).
1.2.3 Sertoli cell function regulation
Sertoli cell function is regulated by an interaction of endocrine and paracrine
signals in vivo. In males, the pituitary gland gonadotropins, follicle stimulation hormone
(FSH) and luteinizing hormone (LH), regulate reproductive functions, such as
spermatogenesis and steroid hormone (estrogen and androgen) production (Figure 1B).
FSH binds to the FSH receptor (FSHR) on the Sertoli cell surface and initiates
spermatogenesis. LH binds to the luteinizing hormone receptor (LHR) on Leydig cells,
where testosterone are produced and released. Testosterone diffuses into the
seminiferous tubules, which in turn stimulates Sertoli cells to produce androgen-binding
protein (ABP) by which the testosterone is concentrated and enriched to promote the
spermatogenesis (McGuinness et al., 1994).
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Figure 2 FSH stimulated cAMP-PKA signaling pathway (Figure 2A) and localization of PKA Cα in
①
different cell types of a seminiferous tubule
in the
Database
. testis of a 120d-old wild-type mouse (Figure 2B1) and
in TM4 Sertoli cells (Figure 2B2-2B3). Figure 2A contains the integrated information from the literatures
(Hunzicker-Dunn and Maizels, 2006; Kim et al., 2005; Lazennec et al., 2000; Misra et al., 2002; Wahli et
al., 1995; Walker and Cheng, 2005) and KEGG Pathway Database①. In Figure 2B1, vimentin (red) was
used as Sertoli cell marker (Guo et al., 2015; Nenicu et al., 2007; Skinner and Griswold, 2004). PKA Cα
subunit distribution exhibits strong differences between different spermatogenic stages of germ cell
development. It is highest abundant in the cytoplasm of round spermatids (rSpt) and elongated spermatids
(eSpt), whereas the cytoplasm of spermatocytes (Spc) contains much less PKA Cα. Spermatogonia (Spg)
are only very weakly labelled by PKA Cα. Sertoli cells (SC) exhibit an intermediate PKA Cα staining
level between spermatocytes* and round spermatids*. PKA Cα is abundant in the cytoplasm and is
present also on a few organelle-like structures in TM4 Sertoli cells, suggesting that FSH-signalling
should work properly in this cell type (Figure 2B2-2B3).
①
http://www.kegg.jp/keggbin/highlight_pathway?scale=1.0&map=map04024&keyword=FSH%20%20AMP%20PKA
Page | 11
Gonadotropin hormones modulate the Sertoli and Leydig cells via the cAMP
signal-transduction pathway: FSH and LH bind to surface receptors on Sertoli or Leydig
cells respectively and thereby stimulate the adenylyl cyclase (AC), causing an increase
in intracelluar levels of cAMP. An increase in intracellular cAMP results in the
activation of the cAMP-dependent protein kinase A (PKA) (Figure 2A), leading to
typical cell-specific responses in both cell types (McLean et al., 2002).
The complex cyclical interplay of FSH and testosterone testosterone maintain
Sertoli cells function by providing the energy required for male germ cell maturation
and testicular structure development. FSH and testosterone play a role in the regulation
of the synthesis of lactate, transferrin, androgen binding protein, transforming growth
factor (TGF) -α and -β, insulin-like growth factor-1 (IGF-1), fibroblast growth factor
(FGF), epidermal growth factor (EGF), mullerian-inhibiting substance (MIS), and
inhibin in Sertoli cells (Skinner and Griswold, 2004).
1.2.3.1 FSH signaling
FSH acts on Sertoli cells by binding the FSH receptor (FSHR). FSHR is a cellsurface receptor containing 7 transmembrane domains. Its structure is very similar to the
LH receptor (LHR) and the thyroid-stimulating hormone receptor (TSHR). FSHbinding promotes the FSHR associated partner, the inactive heterotrimeric G proteins
with α-, β-, and γ-subunits, to release the Gα-GTP, which will bind to the adenylate
cyclase (AC). The adenylate cyclase is then activated to convert ATP into cAMP, which
is an ubiquitous second messenger for intracellular signal transduction. cAMP works by
activating protein kinase A (PKA) to dissociate its C subunit (Cα, Cβ or Cγ), which will
phosphorylate nearby substrates (Lazennec et al., 2000).
One PKA target is the transcription factor CREB (cAMP response element
binding protein). It is rapidly activated by phosphorylation of Ser133 in response to FSH.
Several genes involved in spermatocyte survival are controlled by CREB, such as
lactate dehydrogenase (LDH-A), stem cell factor, aromatase, and IGF-1. Mutation in
CREB can cause more than 75% of spermatid loss due to apoptosis (Scobey et al., 2001;
Walker and Cheng, 2005).
CREB plays also a role in ROS toxicity and mitochondrial biogenesis. These
processes are under the control of the key effector- Peroxisome proliferator activated
receptor gamma coactivator -1α (PPARγ coactivator -1α, PGC-1α). PGC-1α interacts
Page | 12
with CREB as well as the nuclear receptor PPARγ, driving the expression of ROS
detoxifying enzymes, such as GPx1 and superoxide dismutase 2 (SOD2), to regulate
oxidative stress (Figure 2A) (Lee et al., 2009). Interestingly, PPARγ is also a key
regulator of peroxisome metabolism and proliferation (Colasante et al., 2015).
In addition to the transcriptional control induced by FSH/PKA signaling, Akinase anchoring protein (AKAP) might tether the PKA to organelle surfaces on which
the PKA can directly influence metabolic processes, such as by the phosphorylation of
StAR after binding to AKAP1 on the mitochondrial surface (Dyson et al., 2008; Merrill
and Strack, 2014).
1.2.3.2 Testosterone signaling
The androgen testosterone is essential to maintain normal spermatogenesis. A
lower intratesticular testosterone concentration would dramatically reduce the rate of
spermatogenesis, for example testosterone reduction from 20 to 10ng/ml in adult rats
induced an approximately 50% reduction of maintained spermatids per testis (Zirkin et
al., 1989). However, compared with our knowledge on the FSH signaling pathway, little
is known about testosterone-regulation on gene expression in Sertoli cells. Walker and
Cheng (2005) showed that when testosterone is taken up by Sertoli cells through the
plasma membrane, the Pem transcription factor is initiated via the binding of the
androgen receptor (AR) to the androgen response elements (ARE) in the promoter
region to support spermatogenesis (Figure 2A) (Walker and Cheng, 2005).
1.3 AKAPs: Scaffolds for local signaling
AKAP is an acronym for “A-Kinase Anchoring Protein” due to their ability to
bind PKA (Carr et al., 1992). Originally AKAPs were named by their apparent
molecular mass in SDS-PAGE. However, this nomenclature could be confusing when
naming splice variants of the same Akap gene (e.g. D-AKAP1 and S-AKAP84) or
homologs from different species (e.g. human AKAP149 and mouse/rat AKAP121)
(Wong and Scott, 2004). Therefore, the human genome organization nomenclature
committee (HGNC) renamed existing AKAPs from AKAP1 to AKAP14 ① with no
regard to their order of discovery or reference to their traditional name. For example, the
first AKAP discovered by Carr and his colleagues in 1992 is human AKAP79 (Carr et
①
http://www.genenames.org/genefamilies/AKAP
Page | 13
al., 1992), together with its homologs bovine AKAP75 and mouse/rat AKAP150 are
now known as AKAP5. Some AKAPs which were not included in this classification
still maintained their original name, such as Gravin, Ezrin, Rab32 and WAVE-1 (Smith
et al., 2006; Welch et al., 2010). Most AKAPs are usually high-molecular-weight
proteins (e.g. AKAP450 and AKAP-Lbc, each > 300kDa) but some AKAPs exist as
small molecular weight proteins (e.g. GSKIP is only 15.6kDa) (Skroblin et al., 2010).
1.3.1 AKAP binding features and signaling cross-talk
AKAPs bind PKA through the unifying motif of a 14-18 amino acid α-helix
(Day et al., 2011). PKA is normally an inactive heterotetrameric holoenzyme, consisting
of two catalytic and two regulatory subunit dimers (C2R2). Because the majority of
AKAPs prefer anchoring PKA RII subunits dimers, this binding domain was also
termed RII-binding domain (RIIBD). Additionally, AKAPs also contain unique proteinlipid or protein-protein targeting domains for anchoring its complex to specific
subcellular locations (McConnachie et al., 2006).
Members of the AKAP protein family act as unique assemble scaffolds,
tethering PKA and other enzyme components together and directing them to a particular
subcellular location close to the downstream substrates. This spatial separation between
effector and substrate is critical for precise regulation and facilitation of signal
transduction (McConnachie et al., 2006). As shown in Figure 3, all the cAMP-depended
signal transduction pathways work with a series of similar components, such as GPCR,
adenylyl cyclase, protein phosphatase, PDE, Epac and PKA. The isoform of binding
partners recruited by AKAPs depend on the specific cell type. The adenylyl cyclase
generates cAMP; while, PDE degrades it into 5’-AMP, not only determines the level of
cAMP in cells, but also terminates the signal transduction at specific cellular sites.
(Skroblin et al., 2010).
Page | 14
Figure 3 AKAPs assemble the key enzymes and factors, forming a complex to facilitate integration of
cAMP and relative signaling pathways. This picture is taken from the book “International review of cell
and molecular biology” (Skroblin et al., 2010). All the partners have defined functions: G-proteincoupled receptor (GPCR) initiates the signal from external stimulation; adenylyl cyclase (AC) transfers
the external signal into second messenger cAMP; phosphodiesterase (PDE) is responsible for cAMP
degradation, regulates cAMP concentration together with AC; exchange proteins directly activated by
cAMP (Epac) acts as cAMP-effected GTP exchange factors; cyclic nucleotide gated ion channel (cNGC
or CNG) is also a cAMP effector; protein kinase A and other kinases activated by cAMP phosphorylate
multiple associated substrates, which are helpful for the integration and synergy of further signaling
pathways; and protein phosphatase (PP) is mediating the desphosphorylation of those substrates,
avoiding dysregulation of signal transduction.
AKAPs do not only coordinate cAMP signaling, but also regulate other events
by forming multi-functional complexes with protein phosphatases, ion channels, PKD
and other kinases, small GTP-binding proteins, ERK1/2, GSK3β (see subsection
1.4.1.1), IQGAP1 (see subsection 1.4.1.3), StAR or mRNA (see subsection 1.4.2.3).
Therefore, AKAPs play an essential role in integrating the signaling cross talk
(McConnachie et al., 2006; Skroblin et al., 2010).
1.3.2 Structure of AKAP-PKA interaction
AKAP family members contain diverse amino acid sequences without any
conserved region, even the unifying RIIBDs only share less than 30% amino acid
identity (Figure 4). However, despite their sequences diversity, the RIIBDs of AKAP
proteins display a conserved unique structural feature throughout the family, which is an
amphipathic α-helix motif, interacting with D/D domain of the PKA R subunits (Figure
5).
Page | 15
Figure 4 Alignment of the PKA RII-binding regions of the indicated AKAPs and artificial disruptor
peptides (Gold et al., 2006; Hundsrucker et al., 2010; Skroblin et al., 2010)
AKAP RIIBDs consist of hydrophobic and hydrophilic residue pairs, forming a
consensus
pattern
[AVLISE]-X-X-[AVLIF]-[AVLI]-X-X-[AVLI]-[AVLIF]-X-X-
[AVLISE] (X = any amino acid) (Hundsrucker et al., 2010; Skroblin et al., 2010). The
isoelectric point (pI) of known RIIBDs is in the range of 3.43-6.23. The conserved
sequence motif and the pI range could be used together to screen for unknown AKAPs
with low false positive hits ratio in a database.
The PKA holoenzymes are divided into two categories: type-I with RI subunits
(RIα or RIβ) and type-II with RII subunits (RIIα or RIIβ). Type-I PKA is found
primarily in the cytoplasm, whereas the type-II is associated with particulate subcellular
fractions (Dell’Acqua and Scott, 1997). According to the bias of binding PKA R
subunits, AKAPs can be subdivided into 3 classes: RI-, RII-, or dual-specific AKAPs.
For example, AKAP1, AKAP4, AKAP10 and AKAP11 are dual-specific and anchor
both RI and RII subunits (Day et al., 2011; Nipper et al., 2006). In general, the specific
interaction between RIIBD and RII has higher affinity and a lower release-rate than the
interaction between RIIBD and RI subunits. Thus, two peptides were developed as
isoform-selective R-binding peptides: RI-anchoring disruptor (RIAD) (Carlson et al.,
2006) and AKAP-IS which is RII-preferring (Gold et al., 2006). The artificial disruptor
peptides maintain higher affinity and specificity than natural AKAPs. These peptides
bind PKA R dimer by mimicking the RIIBD amphipathic helix structure, so that they
Page | 16
can competitively disrupt PKA-AKAP interaction and inhibit PKA-dependent signaling
by mislocalization of PKA (Carlson et al., 2006).
Figure 5 Crystal structure (PDB ID: 2HWN) displaying the contact point between the AKAP RIIBD
and PKA RII D/D domain (dimerization and docking domain). This picture was generated by Swiss
PDBViewer version 4.1.0. Residues Ile3, Lie5 and Leu21 in D/D domain are necessary for AKAPs/PKA
binding. PKA RII dimer I is formed by helix I (cyan)-turn-helix II (green), and dimer II by helix I’
(pink)-turn-helix II’ (red). Dimer I interacts dimer II in an antiparallel manner resulting an X-form, 4helix bundle, which is called D/D domain. The connection is stabilized by the interaction between the
hydrophobic core of D/D domain and amphipathic helix from AKAPs (Kinderman et al., 2006).
1.3.3 Diversity and evolution within the AKAP family
Most AKAPs are “canonical AKAPs” and present widely in all organ and cell
type, interacting with the D/D domain of PKA R subunit dimers via an amphipathic
helix and anchoring PKA and other partners to target organelles, like the plasma
membrane, the cytoskeleton, mitochondria, the Golgi apparatus, the nucleus, or
vesicular structures (Wong and Scott, 2004).
Some AKAP-PKA binding mechanism work differently. These exceptions are
called “non-canonical AKAPs”, such as pericentrin (Diviani et al., 2000) and inactive
RSK1 (Gao et al., 2010). Their PKA binding sequence could even be a 100-amino acid,
Page | 17
non-helical, Leucine-rich region that interacts with RII. This binding is also insensitive
to AKAP-PKA disruptor peptides. Therefore, the non-canonical AKAPs are often
undetected during AKAP screenings and as such often classified as false positive hits,
or defined as “putative AKAPs”.
Homologues of many mammalian AKAPs were found in lower vertebrates too.
Such as the GSKIP homologue in the one of most basal chordate subphylum, Florida
lancelet (Branchiostoma floridae), named DUF727, which shares more sequence
similarity with human GSKIP than invertebrate GSKIP. There is a big gap between the
vertebrate AKAPs and invertebrate orthologs. For example, all the vertebrate AKAP
GSKIPs bind PKA RII subunit, in contrast to invertebrate or fungal GSKIP homologues
that do not bind RII, suggesting that the RIIBD was formed as vertebrate evolved
(Hundsrucker et al., 2010).
1.4 A closer look to four AKAPs used in this study
As mentioned above in subsection 1.2.3 (Figure 2A), the hypothalamic-pituitarytesticular axis is critical for the male reproductive system development by secreting the
hormone FSH and LH to maintain normal spermatogenesis. Moreover, the cAMP-PKAdependent signaling cascades initiated by FSH and LH stimulation are assumed to be
regulated by certain AKAPs. AKAP11, AKAP1, AKAP4 and AKAP10 have been
identified in the testis and are assumed to contain potential organelle targeting motif.
Therefore, understanding the connection between these 4 AKAPs and the FSH/LHstimulated phosphorylation pathways are necessary to further our knowledge of male
fertility.
1.4.1 AKAP11 (=AKAP220)
AKAP220, now named AKAP11, was first described as an ubiquitously
expressed 220kDa PKA anchoring protein, encoded by the Akap11 gene, which was
previously shown to sequester PKA to peroxisomes (Lester et al., 1996). AKAP220
might influence peroxisomal metabolism in the testis by binding PKA to the
peroxisomes. Therefore, it is of interest to define why male patients with peroxisomal
dysfunction exhibit male infertility and why peroxisomes are very abundant in Sertoli
and Leydig cells (Dastig et al., 2011; Nenicu et al., 2007).
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1.4.1.1 Sequence structure and partner binding sites of AKAP11
The human AKAP220 [Q9UKA4.1] is the most well studied AKAP220, also a
dual-specificity AKAP that can bind both PKA RI and RII subunits (Reinton et al.,
2000). Other binding partners that have been identified by immunoprecipitation include
protein phosphatase 1 (PP1) and glycogen synthase kinase-3β (GSK-3β) (Logue et al.,
2011a; Tanji et al., 2002). The PKA holoenzyme recruitment could enhance protein
phosphatase 1 inhibition, so that the intermolecular interactions influence the other
binding partners’ activity within one signaling scaffold (Schillace et al., 2001). For
example GSK-3β, another enzyme that associates with AKAP220, is phosphorylated by
PKA at Ser9, thereby decreasing its activity. In contrast, protein phosphoatase1
dephosphorylates Ser9 of GSK-3β to enhance its function (Tanji et al., 2002). GSK-3β is
a Ser/Thr kinase that regulates glycogen synthase activity (Plyte et al., 1992), and also
induces apoptosis in cerebellar granule neurons (Li et al., 2000).
PP1 binding motif (PP1α, PP1β and PP1γ)
1
501--505
1195--1198
RII binding site
1901
1650--1663
hAKAP220 [Q9UKA4.1]
1-----------------508
IQGAP1 binding region
1098-------1186
AQP2 binding region
1711----------------------------------- 1901
PP1 inhibitory site
1017-------------------------------------- 1316
GSK-3β binding region
Figure 6 Schematic diagram of hAKAP220 (1901-amino acid) and its partner-binding positions
examined by experiments. The color boxes indicate the binding sites or regions as described with
corresponding color text (Logue et al., 2011b; Okutsu et al., 2008; Schillace and Scott, 1999; Schillace
et al., 2001; Tanji et al., 2002).
1.4.1.2 Subcellular localization
In rats, the first AKAP220 [Q62924.1] was shown by immunofluorescence to be
targeted to peroxisomes, which contains a putative peroxisomal targeting signal (PTS1)
at its C-terminus (the last 3 amino acids “CRL”) (Lester et al., 1996). However, this
peroxisomal targeting remains to be dubious since other researchers have claimed it
might be the result of a “partial overlap of a couple of puncta, possibly caused by
chance, was seen in only a few cells either stained for PMP70 or stained for proteins
with another peroxisomal targeting sequence –SKL” (Day et al., 2011). PMP70 is a 70-
Page | 19
kDa peroxisomal membrane protein. Another AKAP220 isoform [NP_036905.2] in rats
without PTS1 signal was found as the most abundant AKAP in kidney. Double
immunofluorescence staining clearly colocalized AKAP220 with Aquaporin 2 (AQP2)
in the subapical cytoplasmic region of inner medullary collecting ducts (Okutsu et al.,
2008; Uawithya et al., 2008).
The mRNA of hAKAP220 was found to be highly transcribed in human testis,
especially
in
isolated
pachytene
spermatocytes
and
round
spermatids.
Immunofluorescence labeling showed that endogeneous hAKAP220 displayed different
subcellular localizations during the human sperm development: cytoplasmic in
premeiotic pachytene spermatocytes and centrosomal of postmeiotic male germ cells,
elongating spermatocytes and mature sperm. Moreover, the hAKAP220 complex
together with PKA RI, RII and protein phosphatase 1 was suggested to associate with
cytoskeletal structures of the sperm tail (Reinton et al., 2000). hAKAK220 [Q9UKA4.1]
also contains no peroxisomal targeting signal.
In mice, AKAP11 is located in the cytoplasm, and it recruits the type I PKA
holoenzyme. Under the stimulation of increasing cAMP level, PKA C subunits are
released and promote AKAP11 to target free RIα to multivesicular bodies (MVBs). No
colocalization was observed with lysosomes, mitochondria, or peroxisomes, and this
mAKAP220 also doesn’t contain PTS1 signal (Day et al., 2011).
1.4.1.3 Metabolic functions of AKAP220
AKAP220 has been proposed to be involved in the regulation of the urine
concentration. When the animal is dehydrated, the pituitary gland produces arginine
vasopressin (AVP), a peptide hormone that acts on the basolateral plasma membrane of
collecting ducts of the kidney. The AVP targets the vasopressin type 2 receptor (V2R),
thereby initiating a cAMP-dependent and AKAP220-mediated PKA signal cascade in
the collecting ducts. Then the PKA C subunit is activated to phosphorylate a water
channel Aquaporin 2 (AQP2) at Ser256, inducing AQP2 to translocate from cytosol to
the apical membrane. With the increasing number of AQP2 inserted in the apical
membrane, water permeability of the collecting ducts will be enhanced to enable water
reabsorption (Okutsu et al., 2008).
Other research has proposed AKAP220 to be involved in migration of human
fibrosarcoma cells. AKAP220 assembles the cytoskeletal scaffolding protein IQGAP1,
Page | 20
which plays a role in cytoskeletal polarization and cell motility. AKAP220 supports the
spatial and temporal synchronization to regulate dynamic instability of microtubule.
Therefore, AKAP220 could be essential in microtubule dynamic for cancer cells
migration and invasion as shown in Figure 7 (Logue et al., 2011b).
Figure 7 Schematic diagram of AKAP220 complex leading function in cell migration. This picture is
taken from (Logue et al., 2011b). IQGAP1 supports Ca2+/CaM-dependent association of factors that
modulate microtubule dynamics. Under the integration of Ca2+ and cAMP signals, AKAP220 signaling
platform was compartmentalized to the substrates at leading edges of migrating cells. Activated GSK-3β,
which is anchored on AKAP220 complex, phosphorylates the plus-end microtubule tracking protein
CLASP2 at Ser533 and Ser537, and inhibits CLASP2 binding to IQGAP1. When the cellular Ca 2+ level
increases, CaM and Rac could release IQGAP1, so that IQGAP1 is then free to bind AKAP220. With an
increasing cAMP concentration, PKA is activated and can phosphorylate and inhibit GSK-3β, resulting
the releasing of activated CLASP2 to anchor IQGAP1.
Research has shown a link between AKAP220 and the development of oral
cancer. The retinoblastoma (RB) protein is a tumor suppressor protein, which prevents
cell growth and its dysfunction can cause several different types of cancers (Murphree
and Benedict, 1984). Rb pathway is known to alter the cell cycle and progression of
early oral premalignant lesions (OPLs). AKAP220 takes part in the Rb pathway by
regulating protein phosphatase 1 activation and RB protein dephosphorylation and the
transcription switch of genes involved in cell proliferation. AKAP220 overexpressed
significantly in oral tumors, suggesting a role in oral carcinogenesis (Garnis et al.,
2005).
1.4.2 AKAP1
A-kinase anchoring protein 1 is also known as AKAP1, PRKA1, AKAP84,
TDRD17, AKAP121 [Mus musculus], AKAP149 [Homo sapiens], D-AKAP1,
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PPP1R43 or SAKAP84①. AKAP1 binds to RI and RII of PKA and anchors them to the
cytoplasmic face of the mitochondrial outer membrane②.
1.4.2.1 Alternative splicing and subcellular locations
Akap1 transcripts are widely expressed in testis, heart, liver, kidney, skeletal
muscle and brain. Diverse Akap1 transcripts are derived from the same gene by
alternative pre-mRNA splicing. A common N-terminal exon (1-572 amino acids in
human) codes both the mitochondrial targeting sequence (1-30 aa) and the PKA binding
helix (347-360 aa). The C-terminal exons are more conserved, coding K homology
(KH) domain and a Tudor domain. The differing sequence features determine the
various subcellular localizations of AKAP1. For instance, S-AKAP84, a short AKAP1
variant, is mainly expressed in male germ cells (Huang et al., 1999) and targeted
selectively to the mitochondrial sheath in the sperm middle piece (Lin et al., 1995). The
first 30 amino acids in the N-terminus of AKAP1 N0 form a hydrophobic helix
followed by positive charges, which are necessary and sufficient to target AKAP1 to the
mitochondrial outer membrane (MOM) (Huang et al., 1999; Ma and Taylor, 2008).
Additionally, the AKAP1 N1 variant identified in the mouse liver was reported to target
AKAP1 to the endoplasmic reticulum and its N-terminus contained additional 33 amino
acids than the AKAP1 N0 variant and may be generated by either alternative splicing
and/or differential initiation of translation (Merrill and Strack, 2014). The negatively
charged Asp31 within the first 33 amino acid residues converts the mitochondrialtargeting signal into both smooth and rough ER-targeting signal consisting of the first
63 residues, without destroying the mitochondrial-targeting signal. Therefore, the
availability of the Asp31 in AKAP1 N1 variant is responsible to determine its ER or
mitochondrial specific targeting (Ma and Taylor, 2008).
①
②
http://www.ncbi.nlm.nih.gov/gene/8165
http://www.phosphosite.org/proteinAction.do?id=7076&showAllSites=true
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1.4.2.2 Sequence features and domains of AKAP1
Tubuling
binding motif
1
PP1 binding domain
10-----25
153--157
KH domain
565 -------------- 613
857
AKAP1_mouse [O08715]
(AKAP121)
1-----------30
Mitochondrial
306-------325
712--------------771
PKA binding site
Tudor domain
targeting sequence
Figure 8 Schematic diagram of mouse AKAP1 (AKAP121) [O08715] (857 amino acids). The color
boxes indicate the binding sites or regions as described with corresponding color text.
Within the PKA binding domain, the RII tethering domains found in the
AKAP121 of rat (303-322 aa) and mouse (306-325 aa) are 80% identical (Bridges et al.,
2006; Feliciello et al., 1998). Spermatid-SAKAP84 binds RIIα and RIIβ (Lin et al.,
1995), and residues 306-325 constitute the RII-binding site (Chen et al., 1997).
The K homology (KH) domain can interact independently or cooperatively with
different regions of the AU-rich RNAs, and various single strand DNA/RNA-binding
proteins contain such KH domain. NMR (nuclear magnetic resonance) measurement
revealed its β-α-α-β-β-α secondary structure① (Figure 9).
Figure 9 Structure of a
KH domain (PDB ID:
2ANR). This picture was
generated using the Swiss
PDBViewer version 4.1.0.
In the downstream of
AKAP1 protein sequence,
a
KH
domain
is
responsible to bind AUrich 3’-UTR of aimed
mRNA (yellow). The KH
domain displays a unique
secondary
structure
consisting of 3 α-helixes
(pink) and 3 β-sheets
(green): β-α-α-β-β-α.
①
http://pfam.xfam.org/family/KH_1
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AKAP121 binds the 3’ untranslated region (3’-UTR) of certain mRNAs over
the KH domain and plays a role in localization, stabilization, and translational
regulation of these mRNAs (Cardone et al., 2002). The KH domain, residues 565-613 in
mouse and 562-610 amino acids in rat AKAP121, is highly conserved (Feliciello et al.,
1998). The phosphorylation of Ser630 in the AKAP149 KH domain is required for its
RNA binding (627RYVS630 is a conserved PKA phosphorylation site), and
dephosphorylation of this residue prevents RNA association (Rogne et al., 2009).
The Tudor domain of AKAP1 consists of five anti-parallel β-sheets with a
barrel-like fold①, recognizes symmetrically dimethylated Arginines, and also possesses
a weak RNase activity (Grozdanov and Stocco, 2012).
The first 30 residues in all AKAP1 isoforms constitute a mitochondrial targeting
motif. High similarity between the mitochondrial anchor domain of NADHcytochorome b5 reductase and N-terminus of S-AKAP84 has been reported (Beasley et
al., 1993). In 2004, a peptide was derived from the mitochondrial anchor domain of
AKAP121,
[Ac-1XKKPLALPGMLALLGWWWFFSRKKX25-NH2
(X=β-Ala)
(AKAPwt). It displays a helical wheel conformation, which might be essential for both
tubulin binding and mitochondrial targeting. This peptide exhibits a strong competing
inhibitory function, preventing AKAP121 anchoring PKA to the mitochondrial outer
membrane (MOM), and in consequence reduces cAMP effects and its further influence
on cell survival (De Capua et al., 2004).
Furthermore, all AKAP1 isoforms (AKAP121, AKAP149, S-AKAP84) contain
a conserved region to interact with cellular microtubules. The tubulin binding motif (1025 aa) and mitochondrial targeting motif (1-30 aa) overlap each other. The tubulin
monomers might promote AKAP1 scaffold to anchor mitochondria (Cardone et al.,
2002).
1.4.2.3 Binding partners of AKAP1 and responding mechanism and functions
Sequence features determine secondary structures of proteins, and the threedimensional folding of proteins supplies molecular interactions domains for proteinprotein, protein-DNA or -RNA, protein-lipid, protein-ion interaction. Due to these
①
http://pfam.xfam.org/family/TUDOR
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domain and interaction, the subcelluar location and function of a specific protein can be
determined and modified.
1.4.2.3.1 AKAP1 binds double-hairpin loop of „mitochondrial“
mRNAs
The KH domain of AKAP1 binds the stable stem-loop structure of pyrimidinerich RNAs (Livigni et al., 2006). The mRNAs of the steroidogenic acute regulatory
protein (Star), superoxide dismutase 2 (Sod2, MnSOD), ATP synthase (F0-f) and
lipoprotein lipase (LPL) contain double-hairpin structures in their 3’UTR (Dyson et al.,
2008; Rogne et al., 2009). When the KH domain is phosphorylated by PKA, the
AKAP1-mRNA binding stabilizes these mRNAs, leading to more translated proteins
and thereby increasing the abundance of the corresponding proteins in mitochondria
after their import into the organelle (Ginsberg et al., 2003; Grozdanov and Stocco,
2012).
Figure 10 An illustration of cAMP-PKA pathway mediated transcriptional and translational regulation
of StAR. Tropic hormone initiates cAMP-PKA depended signaling pathway to induce Star
transcription. AKAP1 (AKAP121) recruits Star mRNA to the outer mitochondrial membrane,
accelerating the translation and activation of StAR protein. AKAP1 also associates type II PKA to the
mitochondria, which activates StAR at the mitochondrial membrane through the phosphorylation of
Ser195. Some other different kinases also take part in the StAR phosphorylation. And the precursor
37kDa StAR was cleaved into 30kDa mature form, translocating cholesterol from mitochondrial outer
membrane to inner membrane. But this whole processing mechanism is still unclear. This picture is
taken from the review on StAR regulation (Manna et al., 2009).
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1.4.2.3.2 Binding through a direct protein-protein interaction
AKAP1 anchors both PKA RI and RII subunits to the outer mitochondrial
membrane (Herberg et al., 2000). AKAP1/RIα interaction was suggested to influence
the release of neurotransmitter (Perkins et al., 2001).
Human AKAP1 (AKAP149) was reported to bind protein phosphatase 1 via
153
KGVLF157 or
627
RYVSF631 motif (Bridges et al., 2006). Protein phosphatase 1 is a
Ser/Thr phosphatases, consists of one catalytic subunit and one regulatory subunit. Most
protein phosphatase 1 regulators contain an RVXF motif which interacts with the
hydrophobic region of protein phosphatase 1 (Rogne et al., 2009).
AKAP1 also associated with type 4 phosphodiesterases (PDE4), which
influenced T cell activation, and suppressed inflammation by hydrolyzing cAMP
(Asirvatham et al., 2004).
AKAP1 has been shown to interact with a complex of protein tyrosine
phosphatase D1 (PTPD1) (Møller et al., 1994) and the non-receptor tyrosine kinase Src
(Dagda et al., 2011). Src was targeted to mitochondria and activated via PTPD1,
increasing mitochondrial respiration (Livigni et al., 2006). Subsequently, the
mitochondrial Src phosphorylates cytochrome c oxidase (COX) and stimulates COX
activity, which positively regulates components of the respiratory chain (Carlucci et al.,
2008).
A key-signaling event in the control of the mitochondrial morphology is the
reversible phosphorylation of the fission protein dynamin-related protein 1 (Drp1). PKA
phosphorylates Ser637 in human Drp1 (Ser656 in rat Drp1 isoform 1 (Dagda et al., 2011),
results in Drp1 inhibition (Cribbs and Strack, 2007), and therefore increases the
mitochondrial stability and cell survival. AKAP1 also recruits the calcium-responsive
phosphatase calcineurin (CaN, PP2B). CaN dephosphorylates Ser637, thus activating
Drp1 and promoting mitochondrial fission and fragmentation (Carlucci et al., 2008;
Merrill and Strack, 2014).
c-myc binding protein (MYCBP, also called AMY-1) was found to bind to the
PKA RII binding region of AKAP149 and S-AKAK84. AMY-1 and S-AKAP84
mRNAs were found to be strongly expressed in the testis after the appearance of
spermatocytes (Furusawa et al., 2001).
Page | 26
AKAP149 binds to the RNase H region of HIV-1 reverse transcriptase (HIV-1
RT), through the association between residues 375-645 of APAK149 and residues 464541 of HIV-1 RT p66 (Lemay et al., 2008; Warren et al., 2009).
AKAP1 also binds AKAP1 itself to form an AKAP1-dimer via their C-terminal
KH and Tudor domains. Moreover, this self-association requires RNA binding to the
KH domain, but the molecular mechanism and physiological role of AKAP1 selfassociation are not clear (Merrill and Strack, 2014; Rogne et al., 2006).
Figure 11 A model illustrating of AKAP1 scaffold complex on the mitochondrial outer membrane,
assembling diverse mRNA and protein partners to facilitate and integrate signaling pathways as
described above (Merrill and Strack, 2014)
1.4.2.4 Akap1 transcript and posttranslational regulation
The translational coactivator PPARγ (Peroxisome proliferator-activated receptor
γ) was shown to be involved in the Akap1 mRNA regulation. Knock-in mice expressing
mutated PPARγ (P465L), a mutation associated with severe dyslipidemia in humans,
display reduced AKAP1 expression, whereas obese humans treated with the PPARγ
agonist rosiglitazone show increased AKAP1 mRNA levels indicating a possible
involvement of AKAP1 in lipid metabolism (Rodriguez-Cuenca et al., 2012).
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Caspase (Figure 11, Casp) cut the site DSVD of AKAP1 (579-582 aa in
AKAP149). This cleavage results in an N-terminal fragment that contains the
mitochondrial targeting sequence and the PKA binding site and a C-terminal fragment
with the KH and Tudor domains. Whether this caspase cleavage of AKAP1 is
functionally involved in the apoptotic program is unknown (Yoo et al., 2008).
Ischemic insults were shown to promote ubiquitination and degradation of
AKAP1. This occurs through the activation of the transcription factor HIF-1α (Hypoxia
induced factor 1α), which induces Siah2 (E3 ubiquitin-protein ligase 7 in-absentia
homolog 2) for ubiquitination of AKAP1. Ubiquitination accelerates the degradation of
AKAP1 and promotes apoptosis (Kim et al., 2011). Hypoxia induces transcription and
accumulation of Siah2, a RING domain E3-ubiquitin ligase. Siah2 targets PHD1 and
PHD3 enzymes for proteasomal degradation, enabling accumulation of HIF-1α and
downstream activation of hypoxia-induced genes. Hypoxia promotes Siah2-mediated
ubiquitination and proteasomal degradation of AKAP121. This regulatory system,
allows a fast adaptation of ischemic tissue to low oxygen and metabolite levels, and
might be more rapid than transcriptional regulation (Carlucci et al., 2008).
1.4.3 AKAP4
A-kinase anchoring protein 4
①
(AKAP4, PRKA4, AKAP-4, AKAP110,
AKAP82, mAKAP82), acts as a prominent structural protein of the sperm fibrous
sheath (FS), initiating sperm capacitation and influencing sperm motility② (Hu et al.,
2009).
Figure
12
Normal
sperm
structure
(this
picture
is
https://www.studyblue.com/notes/note/n/male-reproductive-system-/deck/4722824)
①
②
taken
http://www.phosphosite.org/proteinAction.do?id=9139&showAllSites=ture
Page | 28
from
Mouse AKAP4 (mAKAP82) and its homologue human AKAP4 (hAKAP82),
are the most abundant proteins of the sperm fibrous sheath as shown in Figure 12
(Brown et al., 2003; Hu et al., 2009). Their precursors, pro-mAKAP82 and prohAKAP82 were also found in the proximal portion or entire length of the principal
piece (Turner et al., 1998). The fibrous sheath is an unique and major cytoskeletal
structure in the principal piece of the flagellum, together with outer dense fibers (ODFs)
and the axoneme, which is required from sperm motility (Nipper et al., 2006). cAMPdependent phosphorylation/dephosphorylation events are critical for the initiation and
maintenance of sperm flagellar beating. mAKAP82 and hAKAP82 localize to the
fibrous sheath, influencing sperm motility by controlling cAMP-PKA signal
transduction. A high homology of sequences was shown among pro-mAKAP82, prohAKAP82 and a 75kDa rat fibrous sheath protein, suggesting conserved structure and
function throughout AKAP4 family (Turner et al., 1998). The Akap4 transcript is highly
expressed during early spermatid development, whereas translated AKAP4 associated
with fibrous sheath only in the late phase of spermatid development. The mature form
of AKAP4 can bind AKAP3, which is another sperm-specific AKAP (Carr et al., 2001),
as well as Fibrous Sheath Interacting Proteins 1 and 2 (FSIP1 and FSIP2) (Brown et al.,
2003). AKAP4 can bind both RIα and RIIα of PKA, and target them to the microtubular
cytoskeleton by AKAP4-tubulin interaction (Nipper et al., 2006).
1.4.4 AKAP10
A-kinase anchoring protein 10① (AKAP10, PRKA10, D-AKAP2, D-AKAP-2) is
a dual-specific AKAP that binds type I and II PKA (Hamuro et al., 2002). AKAP10 has
a C-terminal PKA binding region with conserved 27-residues (e.g. V565 to Q591 in
human AKAP10 [ABB76684.1]) that are sufficient for PKA binding. AKAP10 also
contains one or two putative RGS domains (regulator of G protein signaling), indicating
its function in signal transduction (Wang et al., 2001).
The endogenous AKAP10 protein displayed various subcellular localizations in
different cell types. For example, a mitochondrial pattern was reported in mouse oocytes
(Webb et al., 2008), mouse myocyte cell line, and rat cardiomyocyte primary cells
(Wang et al., 2001). A cytoplasmic staining for AKAP10 was found in a human colon
cancer cell line (Wang et al., 2001). AKAP10 was also detected near postsynaptic and
①
Page | 29
presynaptic rat neuromuscular junction (NMJ) and near the actin region in rat muscle
(Perkins et al., 2001). However, the targeting mechanism has not been clarified yet been
clarified, while the mitochondrial targeting motif was only assumed to be in the Nterminus of AKAP10 (Wang et al., 2001).
AKAP10 plays a role in human heart rhythm control (Tingley et al., 2007).
Genetic AKAP10 mutations could cause sudden cardiac death (SCD), which is one kind
of unexpected rapid natural death occurring within one hour after the initial
symptoms①.
1.5 The function of AKAP1, AKAP4, AKAP10 and AKAP11 in
genital organs
AKAPs in different organs execute distinct specific functions. For example,
AKAP1 is highly expressed in adipocytes, and was suggested to play a role in the
development of obesity (Merrill and Strack, 2014). AKAP1 in bones may promote bone
growth and mineralization (Alam et al., 2010). AKAP121 (=AKAP1) down regulation
has also been suggested to represent an important event in the development of cardiac
dysfunction inducing heart failure (Perrino et al., 2010). Scaffold protein AKAP1 brings
PKA to the mitochondria outer membrane to protect neurons from injury (Merrill et al.,
2011), highlighting the importance of studying compartmentalized signaling networks
in neurodegeneration and neuroprotection (Dagda et al., 2011). In the following
subsections, the roles of AKAPs in the reproductive system are highlighted.
1.5.1 Steroid hormone biosynthesis
The rate-limiting step of steroidogenesis is mediated by the steroidogenic acute
regulatory protein (StAR) (Dyson et al., 2008). StAR activity facilitates the transfer of
cholesterol across mitochondrial membranes to provide the substrate for the cytochrome
P450 side-chain cleavage enzyme (P450scc; CYP11A1). Inside the mitochondria,
P450scc converts cholesterol to pregnenolone, which is the first steroid formed during
the synthesis of all steroids (Miller and Bose, 2011). AKAP1 regulates
posttranscriptional modification and degradation of StAR, especially the intracellular
levels of Star mRNA (Grozdanov and Stocco, 2012). It is suggested that PKA in
①
http://www.phosphosite.org/proteinAction.do?id=18193&showAllSites=ture
Page | 30
complex with AKAP1 directs the regulation of StAR expression in three ways: 1) PKA
serves to control the Star promoter; 2) PKA enhances the translation of Star mRNA;
and 3) PKA promotes StAR phosphorylation and activation (Dyson et al., 2008;
Gomez-Concha et al., 2011).
1.5.2 Sperm development
S-AKAP84 (unique AKAP1 in sperm) accumulates as spermatids undergo
nuclear condensation and tail elongation (Lin et al., 1995). S-AKAP84 and its splice
variant AKAP121 (=AKAP1), anchor PKAIIα to the cytoplasmic surface of
mitochondria. This anchoring has been suggested to be involved in the translocation of
mitochondria to the site of cytoskeleton assembly in male germ cells. This process
ultimately may modulate the motility and/or the fertilization capacity of spermatozoa
(Feliciello et al., 1998). When human AKAP220 (=AKAP11) is expressed in testis,
AKAP220 switches from granular cytoplasmic pattern to the centrosome in mature
sperm. Two AKAP4 isoforms, AKAP82 and AKAP110, are testis-specific and major
structural proteins of the sperm tail principal piece, throughout the fibrous sheathtransverse ribs and longitudinal columns, and are important for sperm motility (Tasken
and Aandahl, 2004).
1.5.3 Oocyte maturation
Homozygous female AKAP1 knockout mice are subfertile or infertile while
heterozygous females and all genotype of males exhibit normal fertility. AKAPs play
dual role in oocyte maturation: 1) the cAMP-PKA dependent growth arrest of oocytes at
meiosis I prophase needed to be maintained by AKAPs prior to the luteinizing hormone
surge. 2) after the initiation of oocyte maturation, AKAP1 is responsible for this
maturation to proceed normally and irreversibly by sequestering PKA catalyzed
phosphorylation from its targets (Newhall et al., 2006). The mitochondrial localization
of PKA (YFP-linked catalytic subunit of PKA encoded from microinjected mRNA,
YFP-PKAcat) is required as the oocyte grows from 61µm to 68µm diameter. This
localization is continuously detectable throughout oocyte maturation until fertilization.
mRNA levels of both AKAP1 and AKAP10 are markedly reduced in growing oocytes,
and increase in the germinal vesicle (GV) stage, peak during metaphase II and
subsequently decrease in the 2-cell embryos stage, suggesting a pattern consistent with
that observed for the exogenous PKA (YFP-PKAcat) localization (Webb et al., 2008).
Page | 31
1.5.4 Prostate cancer
Prostate cancer (PC) remains the most common male cancer in USA. The risk to
develop PC cancer is increased by male infertility (Tvrda et al., 2015). Compare to the
healthy tissues (normal prostate as negative control, and normal testis as positive
control), AKAP4 protein abundance in PC patients biopsies was found to be increased.
The reason for this aberrant expression pattern is not yet clear. One supportive evidence
is that AKAP4 mediates calcitonin-induced invasiveness in PC tumor cells (Chiriva‐
Internati et al., 2012).
1.6 Peroxisomes and infertility
As mentioned in subsection 1.4.1, AKAP220 in rats was suggested to be present
in Sertoli cell peroxisomes. Peroxisomes are small single-membrane organelles present
in all cell types, protecting cells against oxidative stress and lipid toxicity. This
organelle has been associated with cAMP-responsive events, such as androgen
biosynthesis and testosterone secretion (Lester et al., 1996). Peroxisomes also play an
important role in spermiogenesis, spermatogenesis and lipid metabolism including βoxidation of very long chain fatty acids (VLCFA) and cholesterol synthesis in Sertoli
cells of seminiferous tubules (Lüers et al., 2006; Nenicu et al., 2007). The patients with
peroxisomal biogenesis defects such as Zellweger syndrome, display degeneration in
Sertoli cells and Leydig cells (Nenicu et al., 2007). Peroxisomes in germ cells were
found to be functional in the synthesis of polyunsaturated fatty acids (PUFA) which are
the major component of germ cell phospholipids membrane to protect them against
oxidative stress. Peroxisomes were also suggested to be involved in the degradation of
leukotrienes and prostaglandins from paracrine between germ cells and Sertoli cells
(Dastig et al., 2011).
1.6.1 Metabolic pathways in peroxisomes involved in male
fertility
Functional peroxisomes are essential for normal spermatogenesis and the lipid
homeostasis of Sertoli cells. Peroxisomes protect Sertoli cells against ROS and lipid
toxicity. Sertoli cell-specific Pex5 knockout mice were infertile and exhibited a lipid
accumulation in Sertoli cells (Nenicu et al., 2007). Genetic defects in the peroxisomal
membrane transporter ABCD1 results in dysfunction of peroxisomal metabolic
Page | 32
pathways causing adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) disease.
The patients with these defects exhibited severe testicular disorders, such as an
accumulation of VLCFA, abnormal levels of LH and FSH and vacuolated Sertoli cells,
resulting in poor germ cell maturation and reduced fertility (Dastig et al., 2011; Lüers et
al., 2006; Nenicu, 2010). ABCD1 (also known as ALDP) is an adrenoleukodystrophy
protein/lipid transporter of the peroxisomal membrane and also one of the abundant
transporters in Sertoli cells, which mainly regulates cholesterol side chain oxidation by
peroxisomal β-oxidation pathway 2 (Nenicu et al., 2007). ABCD1-3 transport the
different types of fatty acids such as VLCFA, LCFA, 2-methyl-branched chain fatty
acids, dicarboxylic acids, polyunsaturated fatty acids, leukotrienes and prostaglandins
into peroxisomal matrix (Colasante et al., 2015). Recently, substrates specificities of
different ABCD transporters have been analyzed in detail (Ferdinandusse et al., 2014;
van Roermund et al., 2014; van Roermund et al., 2011).
Figure 13 Brief overview of major metabolic pathways and peroxins (in number) in peroxisomes of the
mammalian testis. This picture is adapted from (Colasante et al., 2015; Fahimi and Baumgart, 1999).
Page | 33
Deficiencies of two key enzymes, dihydroxyacetonephosphate acyltransferase
(DAPAT) and alkyl dihydroxyacetonephosphate synthase (ADAPS), involved in
plasmalogens (PLs) synthesis caused mice spermatogenic arrest and a Sertoli cell-only
phenotype. DAPAT and ADAPS are peroxisomal proteins, which carry a PTS1 and a
PTS2 respectively. Another defect in the peroxisomal protein, glyceronephosphate Oacyltransferase (GNPAT), in mice resulted in atrophic testis (Rodemer et al., 2003).
Multifunctional protein-2 (MFP-2, also known as HSD17B4) is a peroxisomal protein
that involved in two β-oxidation pathways to catalyse both straight chain and branched
chain fatty acids in peroxisomes (Huyghe et al., 2006; Nenicu et al., 2007). MFP-2
knockout mice developed neutral lipids accumulation in Sertoli cells and testis
degeneration. This blocked peroxisomal β-oxidation in Sertoli cells might induce the
seminiferous epithelium disintegration and mice infertility (Huyghe et al., 2006).
Interestingly CCDC33 protein, a cancer/testis antigen expressed exclusively in
testis, is a peroxisomal protein containing a putative type 2 peroxisomal targeting signal
(PTS2) (Kaczmarek et al., 2009). This again suggested the peroxisomal proteins play a
role in spermatogenesis.
1.6.2 Peroxisomal matrix protein translocation and import
machinery
Peroxisomal proteins are translated on free ribosomes within the cytoplasm.
Protein translocation and import depend on the presence of peroxisomal targeting signal
sequences (PTS) in peroxisomal matrix proteins located either at the C-terminus or at
the N-terminus and interact with specific cytoplasmic receptors (Dastig et al., 2011;
Lüers et al., 2006).
The two peroxisomal targeting signals are named PTS1 and PTS2. Most
peroxisomal matrix proteins contain a C-terminal PTS1, which is recognized by the
cytoplasmic receptor PEX5p (PEX5Lp in Figure 13 is the long isoform of PEX5p in
mammals with a tetra-tricopeptide repeat domain). A few peroxisomal matrix proteins
contain the PTS2 signal and are shuttled by receptor PEX7p. The PTS2 motif is mostly
located at the N-terminal or less frequently at an internal region of the protein and
consists of the consensus sequence pattern [RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X[HQ]-[LAF] (where X is any amino acid) (Kaczmarek et al., 2009). Both receptors bind
peroxisomal matrix proteins and transfer them from the cytosol to the docking complex
Page | 34
(PEX13p and PEX14p) on the peroxisomal membrane. However, how the peroxisomal
proteins travel across the peroxisomal membrane and are released into the peroxisomal
matrix is still unclear (Islinger et al., 2012; Schrader and Fahimi, 2008).
1.7 Aims of the thesis
Although it is well known that the FSH stimulated PKA pathway affects
spermatogenesis and Sertoli cell functions strongly, until now only one AKAP220
protein has been identified to be involved in targeted PKA regulation of steroid
biosynthesis in Sertoli cells. This PKA-anchoring protein in the rat appeared to be
restricted to the peroxisome because of its PTS1 at the C-terminus. However, it is
unknown whether PKA is targeted to the peroxisomes and its role in peroxisomes has
not yet been investigated. Peroxisomes function in cellular β-oxidation of fatty acids, as
well as LH-responsive testosterone secretion in Leydig cells (Mendis-Handagama et al.,
1992). New information on A-kinase anchoring proteins and their eventual action on
peroxisomal metabolism will help to understand the role of these organelles in the
normal physiology of the testis and in the molecular pathogenesis of male infertility.
Furthermore, understanding the A-kinase anchoring proteins can eventually provide
insights on coordination of organelles involved in steroidogenesis, such as
mitochondria, the endoplasmic reticulum and peroxisomes. Finally, it is known that
PPARα, involved in regulation of many peroxisome genes, is phosphorylated by PKA.
Therefore, it was hypothesized that the AKAP protein with putative PTS1 signals
transported to the peroxisomes may influence peroxisome metabolic events related to
the biological function of a certain cell type.
Therefore, the main aims of this project were:
-
To analyze whether peroxisome gene transcription is influenced by FSH
-
To analyze whether AKAP220 is really localized in Sertoli cell peroxisomes
-
To characterize A-kinase anchoring proteins with putative C-terminal
“PTS1”
-
To clone these AKAPs and analyze their subcellular location in Sertoli cells
-
To get more insights on their eventual function in Sertoli cells
Page | 35
2 Materials
2.1 Cell line and medium
The Sertoli cell line (TM4) and the medium used for the studies are described in
details in Table 1.
Table 1 Cell line and medium (information from ATCC search and Health Protechtion Agency cell line
data sheet 50004291)
Cell line name
TM4
ATCC® Number
CRL-1715™
Catalogue No.
88111401
Lot No.
00C013
Description
Mouse BALB/c testis Sertoli cell
Growth Mode
Adherent
Culture Medium
Ham’s F12 + DMEM (1:1) + 2mM Glutamin [DMEM/F-12+GlutaMax™-L
(GIBCO 31331)] + 5% Horse Serum (HS) + 2.5% Foetal Bovine Serum
(FBS)
Reactivity
From primary culture of Sertoli cell enriched preparations from normal testis
of a 11-13 day old BALB/c nu/+ mouse. Cells respond to FSH but not LH;
Plasminogen activator secretion is FSH and also retinoic acid.
Preservation
Freeze medium: 90% culture medium + 10% DMSO
Storage temperature: liquid nitrogen vapor phase
2.2 Routine Materials
Routine materials used in this thesis are summarized in Table 2.
Table 2 Routine reagents list
Reagent Name
Company / person
Cat. No.
Application
Follicle-stimulating
hormone (FSH)
Sigma-Aldrich
F8174
PKA activator
Cholera toxin (CT)
Sigma-Aldrich
C8052
Gs protein activator
Forskolin (FSK)
Sigma-Aldrich
F6886
Adenylate cyclase (Ac)
Page | 36
activator
8-Br-cAMP
Sigma-Aldrich
B5386
cAMP analog
3-Isobutyl-1-methylxanthine
(IBMX)
Sigma-Aldrich
I5879
Phosphodiesterase (PDE)
inhibitor
H89 dihydrochloride hydrate
Sigma-Aldrich
B1427
Specific inhibitor of PKA
Cell Lysis Buffer (10×)
Cell Signaling
Technology®
#9803
Used for protein isolation
OptiPrep™ Density
Gradient Medium
Sigma-Aldrich
D1556
Used for cell and organelle
isolation
Immun-Star™ AP Substrate
Bio-Rad Laoratories,
Inc.
170-5018
Western blot substrate,
used for detection
Precision Plus Protein™
WesternC™ Standards
Inc.
161-0376
Protein ladder for Western
blots
Precision Plus Protein™
Standards Dual Color
Inc.
161-0374
Protein ladder for Western
blots
RNase-ZAP™
Sigma-Aldrich
R2020
Cleaning agent for
removing RNase
RNA Marker
Promega
G3191
RNA ladder for gel
electrophoresis
Anti-Digoxigenin
Roche Applied
Science
11 333 062 910
Northern Blot
SP6 RNA Polymerase
Roche Applied
Science
10 810 274 001
Northern Blot
T7 RNA Polymerase
Roche Applied
Science
10 881 767 001
Northern Blot
DIG RNA Labeling Mix,
10× conc.
Roche Applied
Science
11 277 073 910
Northern Blot
Nylon Membranes,
positively charged
Roche Applied
Science
11 209 299 001
Northern Blot
Blocking Reagent
Roche Applied
Science
11 096 176 001
Northern Blot
Dispergierer IKA UltraTurrax® T25 basic
IKA-Werke GmbH &
Co. KG
Z404578
High speed homogenizer
RNase AWAY®
MOLECULAR BIOPRODUCTS INC
7000
For eliminating RNase and
DNA from apparatus and
tools surfaces
Diethyl pyrocarbonate
(DEPC)
Sigma-Aldrich
D5758
Nuclease inhibitor, 0.1%
DEPC water is used for
RNA related work
RNAzol® RT
Sigma-Aldrich
R4533
RNA isolation
RNAlater RNA
Stabilization Reagent
Qiagen
76104
Stabilizing RNA away
from risk of degradation
RNeasy Mini Kit
Qiagen
74104
Purification of highquality RNA from tissues
RNeasy Fibrous Tissue Mini
Kit
Qiagen
74704
RNA isolation kit for heart
and muscle tissue
Superscript™ II Reverse
Transcriptase
Invitrogen
18064-022
Reverse transcription
®
Page | 37
Oligo (dT)12-18 Primer
Invitrogen
18418-012
Hybridizes to the mRNA
poly(A) tail and acts as a
primer for first strand
cDNA synthesis
Wasser AccuGENE®
LONZA
733-1631
Water, molecular biology
grade
10× Taq Buffer, with 15
mM Magnesium
5PRIME
2201240
PCR
Taq DNA polymerase
5PRIME
2900166
PCR
Deoxynucleotide Mix,
10mM each dNTP
5PRIME
2900389
PCR
10× Pfu Buffer
Promega
M776A
Pfu-PCR
Pfu DNA polymerase
Promega
M774A
Pfu-PCR
100mM dATP solution
Invitrogen
P/N 55082
Pfu-PCR
Dpn I
Promega
R623A
A restriction enzyme
which digests methylated
DNA
DNase I, Amplification
Grade
Invitrogen
18068-015
Digests single / double
strand DNA
RNaseOUT™ Ribonuclease
Inhibitor
Invitrogen
10777-019
Inhibits RNase A, RNase
B, RNase C
BamHI
Promega
R602A
Restrict enzyme digestion
EcoRI
Promega
R601A
EcoRV
Promega
R635A
HindIII
Promega
R604A
Sal I
Promega
R605A
SphI
Promega
R626A
XhoI
Promega
R616A
Bovine Serum Albumin
Acetylated (BSA)
Promega
R396A
Buffer B
Promega
R002A
Buffer D
Promega
R004A
Buffer K
Promega
R010A
Universal Agarose
BIOSELL
BS20.46.500
Electrophoresis for
checking DNA size
Agarose LE, low electroendosmosis
Boehringer Mannheim
GmbH
1685651
Electrophoresis for DNA
purification
NucleoSpin® Gel and PCR
Clean-up
MACHEREYNAGEL
REF 740609.50
Gel extraction kit
Subcloning Efficiency™
DH5α Competent Cells
Invitrogen
18265-017
Heat shock competent
bacteria
ElectroTen-Blue
Agilent Technologies
200159
Electroporation Competent
Cells
NucleoSpin® Plasmid
MACHEREYNAGEL
REF 740588.250
Plasmid isolation kit
Page | 38
NucleoBond® Xtra Midi EF
MACHEREYNAGEL
REF 740420.50
EF-Midi plasmid isolation
kit
ViaFect™ Transfection
reagent
Promega
E4981
Transfects DNA into cell
lines with high efficiency
and low toxicity
Poly-L-Lysine
hydrobromide
Sigma-Aldrich
P2636
Poly-lysine coating cover
slip
2.3 Buffer recipes
All buffers used in this thesis are summarized in Table 3.
Table 3 Buffer recipes
Buffer
Stability at °C
Buffer A (for Western blot)
1.5M Tris, 13.87mM SDS, pH 8.8
4°C
Buffer B (for Western blot)
500mM Tris, 13.87mM SDS, pH 6.8
4°C
Separating gel composition
(12% acrylamide gel) (for
Western blot)
8ml 30% acrylamide solution, 2ml ddH2O, 10ml
Buffer A, 130µl 10% APS, 15µl TEMED
Immediately
use
Collecting gel composition
(for Western blot)
1.25ml 30% acrylamide solution, 3.75ml ddH 2O, 5ml
Buffer B, 130µl 10% APS, 15µl TEMED
Immediately
use
5 × Running
Western blot)
(for
123.8mM Tris, 1M Glycine, 17.4mM SDS. Dilute it
into 1× electrophoresis buffer before using
4°C
SDS Laemmli Sample Buffer
(for Western blot)
3.55ml ddH2O, 1.25ml Tris-HCl (0.5M, pH 6.8),
2.5ml glycerol, 2ml 10% (w/v) SDS, 0.2ml 0.5%
(w/v) bromophenol blue
-20°C
10×TBS Buffer (for Western
blot)
100mM Tris, 1.5M NaCl, pH 8.0
4°C
1×TBST (0.05% Tween 20)
(for Western blot)
Solve 100ml 10× TBS Buffer and 0.5ml Tween 20
into 1000ml MilliQ H2O
Room
temperature
Stripping Buffer (500ml, pH
6.8) (for Western blot)
10ml 10% (w/v) SDS, 62.5ml Tris-HCl (0.5M, pH
6.8), 427.5ml MilliQ H2O
4°C
Homogenizing Buffer (cell
lysis buffer for peroxisome
isolation)
250mM Sucrose, pH 7.4; 5mM MOPS, pH 7.4; 0.1%
Ethanol, 1mM EDTA; 1mM 6-aminocaproic acid,
0.2mM DTT, Protease inhibitor mix 1:100
Prepare freshly
OptiPrep™ diluent (Solution
B)
0.25M sucrose, 10mM EDTA, 1% (v/v) ethanol,
100mM MOPS-NaOH, pH 7.2
Immediately
use
Working Solution (Solution
C, 54% iodixanol, ρ = 1.291
g/ml)
90% OptiPrep™, 10% Solution B
Immediately
use
Homogenization
(Solution D)
medium
0.25M sucrose, 1mM EDTA, 0.1% (v/v) ethanol,
10mM MOPS-NaOH, pH 7.2
Immediately
use
(rat
0.65ml Ketamin Ratiopharm (Ketamin 100mg/ml);
0.65ml Rompun (Xylazin 20mg/ml); 0.2ml
4°C
Buffer
Narcotic
solution
Anaesthesia)
Page | 39
Sedostress (Azepromazin 10mg/ml); 8.5ml 0.9%
NaCl. Each 0.1ml Narcotic solution is used for 10g
bodyweight.
Transfer
Buffer
(RNA
gel/nylon
membrane
sandwiches)
3M NaCl and 341.78mM sodium citrate in 0.1%
DEPC water, pH 7.0
Room
temperature
1 × Maleic acid buffer (for
Northern blot)
0.1M Maleic acid, 0.15M NaCl, pH 7.5 (20°C),
sterile filtered
20°C
Basic Buffer (for Northern
blot)
40mM Tris-HCl, pH 7.4; 150mM NaCl
1 week at 4°C
Antibody
Buffer
Northern blot)
(for
1% Blocking reagent (w/v) in Basic Buffer
2 months at 4°C
Blocking
solution
Northern blot)
(for
1g Blocking reagent powder (w/v) in 100ml 1 ×
Maleic acid Buffer
4 weeks at 4°C
Washing Buffer 1 (test)
0.1% Tween 20 in Basic Buffer, pH 7.4
Not recommend
25mM Na2HPO4, 1% SDS, 1mM EDTA, pH 7.2
Not recommend
0.3% Tween 20 in 1× Maleic acid buffer, pH 7.5
Not recommend
0.1× SSC
Not recommend
0.1× SSC, 0.1% SDS
Not recommend
0.5× SSC
Not recommend
0.5× SSC, 0.1% SDS
Not recommend
1×SSC: 0.75M NaCl and 0.75M sodium citrate
4°C
1× SSC, 0.1% SDS
Not recommend
Formamide
Hybridization
Buffer (for Northern blot)
50% formamide (v/v) deionized, 5× SSC, 0.1% Nlauroylsarcosine (w/v), 0.02% SDS (w/v), 2%
Blocking solution (v/v)
4°C
10 × MOPS Running Buffer
(for Northern blot)
0.2M MOPS, pH 7; 50mM sodium citrate, 0.01M
EDTA
4°C
10 × Formaldehyde loading
Buffer (for Northern blot)
1mM EDTA, pH 8; 0.25% (w/v) bromphenol blue,
0.25% (w/v) xylene cyanol, 50% (v/v) glycerol
20°C
6× DNA loading Dye
0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene
cyanol, 30% (v/v) glycerol
4°C
Stripping Buffer (for Northern
blot, but not recommend)
50% deionized formamide, 5% SDS, 50mM TrisHCl, pH 7.5
4°C
H3BO3/Na-tetraborate Buffer
(for coating cover slip)
0.7mM Lysine, 100mM boric acid, 47.5mM Natetraborate
4°C
Washing Buffer
Northern blot)
8
(for
2.4 AKAP plasmids
Different A-kinase anchoring plasmids were selected and a database search
performed to find the appropriate IMAGE clones for purchase (Table 4). A plasmid that
contains partial of Akap11 [NM_012773.2] sequence was used as the template for
Northern blot probes design. The plasmids of Akap1 [NM_009648.2], Akap4
Page | 40
[NM_009651.4] and Akap10 [NM_019921.3] were used for detection primers design
and myc-tagged AKAPs overexpression.
Table 4 Commercial plasmids information
Gene
Symbol
Company
I.M.A.G.E
Vector
Cloning Sites
Antibiotics
Akap11
GenomeCube
IMAGp998B0715541Q
pExpress-1
5s: EcoRV,
Ampicillin
(50µg/ml)
3s: NotI
Akap1
GenomeCube
IRAVp968B01135D
pYX-Asc
5s: EcoRI,
3s: NotI
Akap4
Akap10
GenomeCube
GenomeCube
IRCLp5011A0634D
IRAVp968B05107D
pCRBluntIITOPO
5s: TOPO sites,
pSPORT1
5s: SalI,
3s: TOPO sites
3s: NotI
Ampicillin
(100µg/ml)
Kanamycin
(100µg/ml)
Ampicillin
(100µg/ml)
Page | 41
2.5 Primers
All the primers used in this study are listed in Table 5, including verified ones (*) and new designed ones.
Table 5 List of primers designed and used in this study
Gene
Name
Tageted tamplate
Len.
(bp)
Primer
Lab-ID
Primer Lab-Name
Sequence (5'- 3')
Optimized
Ann.
Temp.
(°C)
28S rRNA
NR_003279
254
G12*
rRNA-For1
CCTTCGATGTCGGCTCTTCCTAT
65
G14*
rRNA-Rev1
GGCGTTCAGTCATAATCCCACAG
D24*
ALDP-For2
GAGGGAGGTTGGGAGGCAGT
D26*
ALDP-Rev2
GGTGGGAGCTGGGGATAAGG
K19*
ABCD2-For3
TGCAAAATTCTGGGGAAGA
K20*
ABCD2-Rev2
TGACATCAGTCCTCCTGGTG
L02*
HAbcd3for1
CTGGGCGTGAAATGACTAGATTGG
L03*
HAbcd3rev1
AGCTGCACATTGTCCAAGTACTCC
ABCD1
ABCD2
ABCD3
ACOX1
ACOX2
ACOX3
Catalase
NM_007435
NM_011994.2
NM_008991.2
465
406
523
NM_015729.3
566
D15*
Acox1-For1
CTGAACAAGACAGAGGTCCACGAA
NM_001271898.1
566
D16*
Acox1-Rev1
TGTAAGGGCCACACACTCACATCT
NM_053115.2
688
D17*
Acox2-For1
CTCTTGCACGTATGAGGGTGAGAA
NM_001161667.1
688
D18*
Acox2-Rev1
CTGAGTATTGGCTGGGGACTTCTG
NM_030721.2
813
D19*
Acox3-For1
GCCAAAGCTGATGGTGAGCTCTAT
D20*
Acox3-Rev1
AGGGGTGGCATCTATGTCTTTCAG
D49*
Catalase-For3
ATGGTCTGGGACTTCTGGAGTCTTC
D53*
Catalase-Rev3
GTTTCCTCTCCTCCTCGTTCAACAC
NM_009804.2
Page | 42
833
67
58
65
60
60
55
64
Gpx1
SOD1
Pex5
Pex13
Pex14
Pex11α
Pex11β
Pex11γ
PPARα
PPARβ
PPARγ
FSHR
NM_008160.6
NM_011434.1
197
153
J64*
Gpx1-For1
GGGACTACACCGAGATGAACGA
J65*
Gpx1-Rev1
ACCATTCACTTCGCACTTCTCA
G40*
SOD1-For1
TGGGTTCCACGTCCATCAG
G42*
SOD1-Rev1
ACACCGTCCTTTCCAGCAG
NM_008995.2
508
F47*
Pex5-For1
GAGTGAAGAAGCAGTGGCTGCATAC
NM_175933.2
508
F48*
Pex5-Rev1
GGACAGAGACAGCTCATCCCTACAA
NM_023651.4
718
F27*
Pex13-For1
GACCACGTAGTTGCAAGAGCAGAGT
F28*
Pex13-Rev1
CTGAGGCAGCTTGTGTGTTCTACTG
F29*
Pex14-For1
CACCTCACTCCGCAGCCATA
F32*
Pex14-Rev1
AGGATGAGGGGCAGCAGGTA
F20*
Pex11alpha-For1
TCAGCTGCTGTGTTCTCAGTCCTT
F19*
Pex11alpha-Rev1
GTACTTAGGAGGGTCCCGAGAGGA
F21*
Pex11beta-For1
ATCTGTCCCTGGCTACGACTCAAG
F22*
Pex11beta-Rev1
AAACTGGAAAGTGTGGAGGCAGTC
F23*
Pex11gamma-For1
GACTCTGCTTGGTGGTGGACACT
F24*
Pex11gamma-Rev1
TGTCTCTCCCACTCACCTTTAGGC
NM_019781.2
NM_011068.1
NM_011069.3
NM_026951.2
131
420
590
682
NM_011144.6
584
F68*
PPAR alpha-For2
AGACCGTCACGGAGCTCACA
NM_001113418.1
584
F70*
PPAR alpha-Rev2
GGCCTGCCATCTCAGGAAAG
NM_011145.3
363
F71*
PPAR beta-For1
CACCGAGTTCGCCAAGAACA
F72*
PPAR beta-Rev1
AGAGCCCGCAGAATGGTGTC
NM_001127330.1
441
F74*
PPAR gamma-For2
TCCGTAGAAGCCGTGCAAGA
NM_011146.3
441
F76*
PPAR gamma-Rev2
CACCTTGGCGAACAGCTGAG
NM_013523.3
345
L70*
Fshr F1
CCAGCCTTACCTACCCCAGT
L71*
Fshr R1
CTGTGGTGTTCCCAGTGATG
61
63
64
65
60
64
63
64
68
60
60
62
Page | 43
GAPDH
NM_001289726.1
E12*
GAPDH-For1
CACCATGGAGAAGGCCGGGG
417
E15*
GAPDH-Rev1
GACGGACACATTGGGGGTAG
60
440
BK16
T7-GAPDH-R
TAATACGACTCACTATAGGGAGAGACGGACACATTGGGGGTAG
60
T7 RNA polymerase promoter
β-actin
NM_007393.3
M29*
Mm beta-Actin1146F
GGATCAGCAAGCAGGAGTACGAT
120
M30*
Mm beta-Actin1266R
CTGCGCAAGTTAGGTTTTGTCAA
62
143
BK17
T7-beta-actin-R
TAATACGACTCACTATAGGGAGACTGCGCAAGTTAGGTTTTGTCAA
62
StAR
NM_011485.4
AKAP11LSP
292
K21*
StAR-For1
GTTCCTCGCTACGTTCAAGC
K22*
StAR-Rev1
TTCCTTCTTCCAGCCTTCCT
AZ47
LSP-1240F
AATGTTAGAAAGCCAACACC
230
AZ48
LSP-1449R
GCATAATCTTCGTAAGTGCAG
56
253
BK18
T7-LSP-R
TAATACGACTCACTATAGGGAGAGCATAATCTTCGTAAGTGCAG
56
NM_012773.2
58
AKAP11BSP1
NM_012773.2
AZ49
BSP-3113F
ATCTGACTAATACTGCGCTTG
308
AZ51
BSP-3399R
CTCAGATAAACTACCAACGGAT
56
331
BK19
T7-BSP1-R
TAATACGACTCACTATAGGGAGACTCAGATAAACTACCAACGGAT
56
AKAP11BSP2
AKAP11BSP3
NM_012773.2
NM_012773.2
Page | 44
893
608
AZ50
BSP-2528F
GCCAGAATGACTTCAAACCAAC
AZ51
BSP-3399R
CTCAGATAAACTACCAACGGAT
AZ50
BSP-2528F
GCCAGAATGACTTCAAACCAAC
AZ52
BSP-3115R
CTCAAGCGCAGTATTAGTCAG
56
57
AKAP11rat
M13
promotor
NM_012773.2
1147
AZ57
rAKAP11-2124F
CTTCACAGCAAAGGCAGCAG
AZ58
rAKAP11-3341R
ATTCTTTGGCTAACTCCCCTT
pGEM-Teasy
plasmid
266
BE61
M13FS
CCCAGTCACGACGTTGTAAAACG
Express 1 plasmid
190
BE62
M13RS
AGCGGATAACAATTTCACACAGG
AKAP1-N
terminus
NM_009648.2
402
BJ25
F1N
CGGAGACAAAGCTATGACCC
NM_001042541.1
295
BJ26
R1N
CTTGCCAATCAGTCGACCA
AKAP1-C
terminus
NM_009648.2
312
BJ27
F1C
TTTGTGACCCTGCCATTCCAA
NM_001042541.1
312
BJ28
R1C
AACAGTGCAAAGAAGACCGAA
AKAP1
IMAGE
clone
IRAVp968B01135D
2573
BH42
AKAP1-Mm-F3B
ATAGGATCCGCAATCCAGTTGCGTTCGCTC
55
60
54
54
58
BamHI
BH43
AKAP1-Mm-R3B
ATACTCGAGTCAGAGGCTGGCATAGTAGCT
XhoI
IRAVp968B01135D
2573
BH48
AKAP1-Mm-F5B
ATAGGATCCATGGCAATCCAGTTGCGTTCG
BamHI
BH49
AKAP1-Mm-R5B
Stop codon
58
Start codon
ATACTCGAGGAGGCTGGCATAGTAGCTGTC
XhoI
IRAVp968B01135D
BL03
1-5B-771F
GGACTGGAGATGCTGTGTTGG
IRAVp968B01135D
BL27
1-5B-1420F
ACCAAGGACCAGAAGCCAAAG
IRAVp968B01135D
BL30
AKAP1-Mut-F
ATGGTATCACTGTGGAAGTCATCGTGGTCAAC
BL31
AKAP1-Mut-R
GGTTGACCACGATGACTTCCACAGTGATACCATC
AKAP4-N
terminus
NM_009651.4
202
BH58
F4N
ACAACAAGATCAGGACCGAA
NM_001042542.2
202
BH59
R4N
GAAAAGGCACACAGATCCCTC
AKAP4-C
terminus
NM_009651.4
224
BH60
F4C
TGGCCCTGAAGTTATTGTCAACA
NM_001042542.2
224
BH61
R4C
TGGCAAACTTCATGACCTCC
55
53
53
Page | 45
AKAP4
IMAGE
clone
IRCLp5011A0634D
2550
BH44
AKAP4-Mm-F3B
ATAGGATCCATTGCCTACTGTGGTACTACA
58
BamHI
BH45
AKAP4-Mm-R3B
ATACTCGAGTTACAGGTTAGCGAGAAGCAA
XhoI
IRCLp5011A0634D
2550
BH50
AKAP4-Mm-F5B
Stop codon
ATAGGATCCATGATTGCCTACTGTGGTACT
58
BamHI Start codon
BH51
AKAP4-Mm-R5B
ATACTCGAGCAGGTTAGCGAGAAGCAAGTC
XhoI
IRCLp5011A0634D
BL05
4-3B-920F
ATTGCTTCGGAGATGGCCCAT
IRCLp5011A0634D
BL06
4-3B-1830R
CACATGGATCTTTGCCCTTGG
BK20
F10C
TCCCTGGCTGCTCACGGCTCT
BK21
R10C
AGCTCTTCTTGGGCCTCGTC
BH47
AKAP10-Mm-R3B
ATACTCGAGTCATAGCTTTGTAGACTTCTC
AKAP10C terminus
NM_019921.3
AKAP10
IMAGE
clone
IRAVp968B05107D
335
1018
XhoI
BL28
10-3B-355F
56
58
Stop codon
GAATTCAAAAGTATAGAACAAGATGCAGTG
EcoRI
IRAVp968B05107D
1018
BH53
AKAP10-Mm-R5B
ATACTCGAGTAGCTTTGTAGACTTCTCTAG
58
XhoI
BL29
10-5B-355F
GAATTCATGAAAAGTATAGAACAAGATGCA
EcoRI Start codon
myc-GFP3B
pGeneClip hMGFP
vector
701
BL48
GFP-BamHI-NoStart
ATAGGATCCGGCGTGATCAAGCCCGAC
BE66*
RvGfP-XhoI
ATACTCGAGTTAGCCGGCCTGGCGGG
XhoI
GFP-myc5B
pGeneClip hMGFP
vector
701
BE65*
fvGfP-BamHI
Stop codon
ATAGGATCCATGGGCGTGATCAAGCC
BamHI Start codon
BE67*
RvGfP-XhoIWoS
ATACTCGAGGCCGGCCTGGCGGG
XhoI
Page | 46
62
BamHI
59
2.6 Antibody list
All antibodies used in this thesis are listed with appropriate source, catalog
number and dilution used in the different experiments (Table 6).
Table 6 Antibody dilution list
Antibody Name
Host
Sourse (company / person)
Catalog ID
Dilution
Anti-Digoxigenin
Mouse
monoclonal
Ab
Roche Applied Science
11 333 062
910
3 : 5000 (NB)
AKAP220 (H300)
Rabbit
polyclonal
Ab
Santa Cruz Biotechnology,
Inc.
Sc-135335
1 : 200 (WB)
Oxphos III core
subunit II
Mouse
monoclonal
Ab
Molecular Probes,
Invitrogen
459220
1 : 5000 (WB)
Phospho(Ser/Thr) PKA
Substrate
Rabbit
polyclonal
Ab
Cell Signaling
Technology®
#9621
1 : 1000 (WB)
PKA C-α
(D38C6)
Rabbit
monoclonal
Ab
Cell Signaling
Technology®
#5842
1 : 1000 (WB);
Catalase
Rabbit
polyclonal
Ab
Gift from Denis I. Crane,
School of Biomol. Biophys.
Sci., Griffith Univ., Nathan,
Brisbane, Australia
/
1 : 500 (IF)
Myc-Tag (9B11)
Mouse
monoclonal
Ab
Cell Signaling
Technology®
#2276
1 : 8000 (IF)
Pex3
Rat
polyclonal
Ab
made by Dr. Claudia
Colasante
/
1 : 200 (IF)
Pex14
Rabbit
polyclonal
Ab
Gift from Denis I. Crane,
School of Biomol. Biophys.
Sci., Griffith Univ., Nathan,
Brisbane, Australia
/
1 : 100 (IF)
Superoxide
dismutase 2
(SOD2, Mn-SOD)
Rabbit
polyclonal
Ab
Research Diagnostics, Inc.,
NJ, USA
RDIRTSODMa
bR
1 : 5000 (IF)
anti-mouse IgG
Alkaline
Phosphatase
antibody
Goat
Sigma-Aldrich
A3562
1 : 30,000 (IB)
anti-rabbit IgG
Alkaline
Phosphatase
antibody
Goat
Sigma-Aldrich
A3687
1 : 30,000 (WB)
anti-mouse IgG
Donkey
Molecular Probes
A21202
1 : 300 (IF)
1 : 400 (IF)
Page | 47
Alexa Fluor 488
green
anti-mouse IgG
Alexa Fluor 555
red
Donkey
Molecular Probes
A31570
1 : 300 (IF)
anti-mouse IgG
Texas Red
Horse
Vector Laboratories
TI-2000
1 : 1000 (IF)
anti-rabbit IgG
Alexa Fluor 488
green
Donkey
Molecular Probes,
Invitrogen
A21206
1 : 500 (IF)
anti-rat IgG Alexa
Fluor 594 red
Goat
Molecular Probes
A11007
1 : 300 (IF)
Page | 48
3 Methods
3.1 Bioinfomatic screening for the AKAPs with putative PTS1 signals
In 1980’s, the Gould and Subramani group started to investigate a peptide
targeting signal that necessary and sufficient to transport proteins into peroxisomes.
Through a series of gene mutagenesis, they demonstrated the firefly luciferase contains
minimal peroxisomal targeting signal consists of three amino acids, Ser (S)-Lys (K)Leu (L), at the extreme carboxy terminus. They also suggested this minimal PTS
tripeptide with consensus sequence was a conserved feature of peroxsomal proteins
through evolution (Gould et al., 1987; Gould et al., 1989). In order to identify proteins
of the AKAP family containing a peroxisomal targeting signal 1 (PTS1), a computer
program was developed to scan all AKAP member sequences for an eventual PTS1
tripeptide.
3.1.1 Scan of AKAP sequences for the consensus PTS1 motif
All known AKAP amino acid sequences were downloaded from UniProt protein
database, including validated and predicted AKAPs (1306 items in all), to produce a test
library. Peroxisomal matrix proteins known to contain a real “PTS1 binding site” were
downloaded from the PTS1 BLOCK① at the “PeroxisomalDB Home” webpage②. Total
98 PTS1 containing proteins from Homo sapiens, Mus musculus, Saccharomyces
cerevisiae and Arabidopsis thaliana are highly conserved in evolution. The PTS1
tripeptide consensus from these 98 proteins was summarized as [A/C/H/K/N/P/Q/S/T][A/H/K/N/Q/R/S]-[F/I/L/M/V]. Within the 1306 AKAPs, the sequence matched by this
motif were selected and saved into a *.txt file (81 items in all).
3.1.2 Use of ClustalX 2.0 & MEGA 4.0.2 programs to create an
evolutionary tree
The AKAPs sequence file needed to be loaded into the ClustalX 2.0 program to
accomplish a complete sequence alignment, and saved as *.dnd and *.aln files.
Thereafter, the program MEGA 4.0.2 was used to convert the *.aln file into a *.meg
file, which is the MEGA format. The new file of the *.meg format can be indicated as
①
②
http://216.92.14.62/diy_PTS1.html
http://www.peroxisomedb.org/home.jsp
Page | 49
“Protein Sequence” by MEGA 4.0.2 and constructed a Phylogeny tree by using the
“Neighbor-Joining” (NJ) algorithm. The program will execute a self-calculation and
generate a tree image, which is in a *.tiff format.
3.1.3 Use of Python 2.7.5 & R i386 3.0.1 programs to test
AKAP1, AKAP4, AKAP10 through PTS1 predictor
From the rough PTS1 screening throughout 14 AKAP families, 3 families of 14
(AKAP1, AKAP4 and AKAP10) were implied to be evolutionary conserved
“peroxisomal” AKAPs. Then the protein members of these 3 families were respectively
further analyzed by 1) a PKA binding domain screening and 2) a comparable strict
PTS1 online prediction.
PKA binding domain forms a consensus pattern: [AVLISE]-X-X-[AVLIF][AVLI]-X-X-[AVLI]-[AVLIF]-X-X-[AVLISE] (X = any amino acid) (Hundsrucker et
al., 2010; Skroblin et al., 2010).
As mentioned in Brocard’s paper, the PTS1 signal is considered to consist of 3
parts: a crucial C-terminal tripeptide, an upstream tetrapeptide, and a flexible
approximately 12 amino acid long hinge (Brocard and Hartig, 2006). Their group also
successfully developed an online tool to predict candidates①. Computer programming
was used for sequence scaning under the Python 2.7.5 environment, and the R i386
3.0.1 program was used to convert the statistical results generated from Python into
ostensive graphs. The codes of Python (in blue frame) and R (in purple frame) are
presented in the end of this thesis (see Chapter 12. Supplemental data S1), and the
screening workflow was set as follows (this workflow of AKAP1 as the example):
①
http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp
Page | 50
Download all the AKAP1 protein sequences from NCBI protein database, and save as a *.fasta file
seq_num_len_des_read-code.py
seq_num_len_des_read.txt
Rename that original *.fasta file as “All AKAP1 protein from NCBI-143 seq.fasta”
Remove_duplicate_seq-code.py
seq_without_duplicate.txt
id_order.txt
AKAP_domain_selection-code.py
AKAP_true_protein.txt
AKAP_true_protein_info.txt
PTS1_predictor_filter-code.py
AKAP_false_protein.txt
AKAP_true_protein_info.xls
PTS1_pred_score.txt
PTS1_pred_score.xls
AKAP_PTS1_for_R.xls
AKAP_PTS1_for_R.txt
Barplot_AKAP1.R
AKAP1_domain_feature.pdf
Page | 51
3.2 Cell culture
3.2.1 Cell culture routine
The procedure was slightly modified from the original protocol from ATCC①. At
first passage, cells which have been resuscitated from a frozen stock were split at a ratio
of 1:3. Thereafter, the culture medium was renewed every 2 to 3 days, until cells were
fully confluent. For subculturing, the cell layer was briefly rinsed with 0.25%
trypsin/EDTA 1ml per 200ml flask and incubated at 37°C for 5 minutes to induce the
detachment. Cells were gently removed and mixed by aspiration and pipetting. The subconfluent cultures were split in a concentration of 1×104 cells/cm2, and incubated at
37°C with 5% CO2.
3.2.2 Cell number and viability examination
The Sertoli cell number was determined with a hemocytometer. Trypan blue
exclusion assays were used to determine the percent survival of the Sertoli cells. The
procedure was slightly modified from the original Sigma “Protocol 6 – Cell
Quantification”②.
The adherent cells needed to be suspended as above described, thereafter, 150µl
of that cell suspension was transferred into an Eppendorf tube under sterile conditions
and mixed gently with 150µl of Trypan Blue. The needed hemocytometer and coverslip
were cleaned by EtOH and ddH2O and air-dried. The chamber was filled with 10µl of
the stained cell suspension without air bubbles. The cell number was counted under
20 × magnification with a bright field/phase contrast-equipped inverted lightmicroscope. Dead cells were recognised by being blue stained. Ideally, >100 viable
cells were used for accurate counting. Finally the cell concentration of the Eppendorf
tube, which was used in the first step before Trypan blue stain, was calculated with the
following formula:
Viable cell concentration =
①
(total live cell numbers from 4 corner squares)÷4×9
3𝑚𝑚×3𝑚𝑚×0.1𝑚𝑚
http://www.lgcstandards-atcc.org/products/all/CRL1715.aspx?geo_country=de#culturemethod
②
http://www.sigmaaldrich.com/technical-documents/protocols/biology/cellquantification.html
Page | 52
×2
3.2.3 Isolation of total RNA from TM4 cells
According to the experiment requirement, TM4 cells were grown in flasks or 6well plates. The whole procedure of total RNA isolation was modified from the Qiagen
protocol “Purification of Total RNA from Animal Cells Using Spin Technology” in
RNeasy® Mini Handbook, 4th Edition. For a 200ml flask as an example, cells were
cultured upto an ideal density, the medium was discarded and 10ml PBS was added for
rinsing. Then the cells were trypsinized by 0.1-0.25% trypsin at 37°C for 10min. 8ml
new medium was added to stop the digestion and the cells were resuspended for the cell
number counting as described above and collected by centrifugation at 300×g for 5min.
After removal of the supernatant, the cell pellet (ideally < 5×106 cells) was resuspended
and disrupted in 350µl Buffer RLT (including 1% β-mercaptoethanol, the volume of
RLT Buffer used depends on the cell number, see page 28 in handbook of Qiagen
RNeasy Mini Kit) and homogenized by spinning through a QIAshredder column,
supplied by the Qiagen RNeasy Mini Kit. The mixture of 10µl DNase I (RNase-free)
and 70µl RDD Buffer (Qiagen) was added onto the column to digest the contaminated
genomic DNA. Thereafter, the rest procedure was done as described in the
manufacturer's instruction.
3.3 RT-PCR
According to the protocol from Invitrogen “First-Strand cDNA Synthesis Using
SuperScript™ II RT” ① , 2µg total RNA (in 10µl) was reverse transcribed in a 20µl
reaction mixture at 42°C for 50min using 200U of SuperScript™ II RT and 500ng of
oligo-dT primers in the 1× first-strand synthesis buffer. The PCR reaction was done in a
volume of 25µl reaction mixture containing 2µl of cDNA (reverse transcription
reaction), 18.8µl ddH2O, 2.5µl of 10× PCR Buffer, 0.5µl of 10mM dNTP Mix, 2µl of
10µM Forward + Reverse primer mixture, 0.2µl of 5U/µl Taq DNA polymerase. The
PCR conditions were first a 5min denaturation period at 95°C and subsequent 35 cycles
of denatureation at 95°C for 30 sec, annealing of individual primer pairs at optimal
temperature for 45 sec, extainsion at 72°C for 1 min and one final extension for 10 min
at 72°C.
①
http://tools.lifetechnologies.com/content/sfs/manuals/superscriptII_pps.pdf
Page | 53
3.4 SDS-PAGE and Western blots
According to the protocol in our lab, a standard curve for protein measurements
was made with the Bradford method with serial BSA gradient dilutions. The adherent
cells were lysed by Lysis buffer (Cell Signaling) and handled following manufacturer's
instruction. Thereafter, the protein concentrations of the samples supernatant were
measured triplicates by using a spectrophotometer at OD595. 50µg of protein samples
per lane were loaded onto 12% SDS-polyacrylamide gel, together with dual color
marker in the 1st lane and unstaining marker in the 2nd lane. The proteins were collected
by electrophoresis at 80V until they reached the separation gel and thereafter separated
at 120V. The separated proteins were transferred from the SDS-gel onto a
polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in 10%
nonfat milk powder at 4°C overnight for blocking of non-specific protein binding sites.
The next day, the milk was removed and the membrane was incubated with first
antibody overnight also at 4°C. On the 3rd day morning, the membrane was washed with
1×TBST 3 times for 10min each at room temperature. The bound primary antibody was
detected by incubation with an alkaline phosphatase-conjugated secondary antibody for
1h at RT. The membrane was washed with 1×TBST 5 times for 10min each at RT.
Immune-Star™ AP was used as substrate to visualize the bands with immunecomplexes
in the dark room. Western blots were exposed from 30s to 10min to X-ray film to
visualize the appropriate bands.
3.5 Peroxisome purification by OptiPrep™ density gradient
centrifugation
OptiPrep™ is a 60% (w/v) solution of Iodixanol® in water, density = 1.32 g/ml,
which is used for density gradient centrifugation to purify subcellular organelles.
Iodixanol is an ideal medium, since it is non-ionic and non-toxic to cells and
metabolically inert. The Iodixanol solusion possesses a low viscosity and can be made
isosmotic at variant densities, which is helpful for a rapid separation of whole cells or
isolated organelles①. It was reported that peroxisomes can be highly purified from other
organelles by this method (Van Veldhoven et al., 1996). In iodixanol, lysosomes band at
①
http://www.axis-shield-density-gradientmedia.com/Purification%20of%20subcellular%20organelles.pdf
Page | 54
a density of 1.115 g/ml, endoplasmic reticulum band at a density of 1.13 g/ml,
mitochondria band at a density of 1.13-1.15 g/ml and peroxisomes band at a density of
1.18-1.20 g/ml (Graham, 2002a; Graham, 2002b).
The isosmotic gradient should be prepared one day before and immediately
frozen at -20°C. The gradient is thawed 15min before usage.
Table 7 Properties and preparation of Iodixanol-sucrose solution
No.
% iodixanol
ρ (g/ml)
Solution C
Solution D
1
18%
1.12
2ml
3ml
2
25%
1.15
1.82ml
1.68ml
3
34%
1.19
4.08ml
1.92ml
4
40%
1.22
2.4ml
0.6ml
5
48%
1.26
3.84ml
0.16ml
In order to get pure peroxisomal fractions from the TM4 cell culture (~107 cells),
differential centrifugation was used after homogenization of the cells. The whole
procedure is depicted in details in Figure 14 A&B.
Page | 55
Collect cells from 4 flasks (200ml),
wash with PBS
1200 rpm for 10min (Thermo
Scientific™ Sorvall™ Heraeus Rotor
75006445)
Take out the supernatant, and weigh the cell pellet
Grinding on ice for 1 min (Potter-Elvehjem homogenizer)
Add 3ml Homogeniation Buffer per 1g cells
Check the cell lysis by Trypan Blue
Take 50µl supernatant into liquid nitrogen, and keep at -80°C, named S1. Centrifuge the rest supernatant at 1,900 ×g for 12min at 4°C
Take 50µl into liquid nitrogen, and keep at -80°C, named H1
S1
H1
Centrifuge the rest at 100 ×g for 12min at 4°C
Take 50µl pellet debris into liquid nitrogen, and keep at -80°C, named P1. And discard the rest of the celldebris
P1
Take 50µl supernatant into liquid nitrogen, and keep at -80°C, named S2. Centrifuge the rest supernatant at 25,000 ×g for 12min at 4°C
S2
Resuspend pellet in 50µl Homogenization Buffer, put into liquid nitrogen, and keep at -80°C, named P2.
P2
Take 50µl supernatant into liquid nitrogen, and keep at -80°C, named S3. Centrifuge the rest supernatant at 29000rpm for 1h at 4°C
(SORVALL® Ultra Pro 80 Refrigerated Ultracentrifuge with Sorvall® AH-629 Swing Buket Rotor)
S3
Take 50µl supernatant into liquid nitrogen, and keep at -80°C, named S4.
S4
Resuspend pellet with enriched peroxisomes in 300µl Homogenization Buffer. Take 50µl into liquid nitrogen and keep at -80°C, named P3.
P3
The rest 250µl was used for OptiPrep gradient centrifugation.
Page | 56
Resuspend pellet in 50µl Homogenization Buffer, put into liquid nitrogen,
and keep at -80°C, named P4.
P4
250µl P3 (light mitochondria + enriched peroxisomes)
Fractions (6 drops / tube × 12 tubes,
named B1 - B12)
(18%) ρ1 = 900 µl
(25%) ρ2 = 630 µl
60,000 ×g for 2h30min
(34%) ρ3 = 540 µl
(40%) ρ4 = 270 µl
(48%) ρ5 = 360 µl
3ml / Tube
(Immediately freeze at -20°C, take it out just 15min before usage)
Figure 14 Schematic illustration of peroxisome purification by OptiPrep™ (keep all solutions and carry out all operations at 4°C).
Page | 57
The fractions were named as follows:
H1: total homogenate;
S1:
supernatant after 1st centrifugation from H1, containing all organelles and cytosol;
P1:
pellet after 1st centrifugation from H1, mainly containing cell debris;
S2:
supernatant after 2nd centrifugation from S1, mainly containing small
mitochondria, peroxisomes, lysosomes, microsomes and cytosol;
P2:
pellet after 2nd centrifugation from S1, mainly containing heavy mitochondria and
nuclei, a few large peroxisomes;
S3:
supernatant after 3rd centrifugation from S2, mainly containing microsomes and
cytosol;
P3:
pellet after 3rd centrifugation from S2, mainly containing enriched peroxisomes,
light mitochondria, few lysosomes and microsomes, used for further OptiPrep
density gradient centrifugation;
S4:
supernatant after 4th centrifugation from S3, mainly containing cytoplasmic
proteins;
P4:
pellet after 4th centrifugation from S3, mainly containing microsomes and very
small peroxisomes.
250µl P3 was then added on top of a discontinuous OptiPrep gradient (3ml 18%48% iodixanol: 900µl 18%, 630µl 25%, 540µl 34%, 270µl 40% and 360µl 48%) in
thick-walled polycarbonate tubes. The gradient was centrifuged at 60,000 × g for
2h30min in a superspeed centrifuge (Thermo Scientific Sorvall® Evolution™ RC).
During centrifugation the gradient became continuous and the peroxisomal fraction
displayed a smeared white suspended layer in the tube. A needle was carefully inserted
and lowered to the tube bottom. All fractions (6 drops per tube) were collected of the
3ml gradient solution by a peristaltic pump into Eppendorf tubes. A total of 12 numbers
of fractions were collected (named B1-B12) in separate tubes for further protein
analysis.
Page | 58
3.6 Rat dissection and RNA isolation
3.6.1 Anaesthesia for rats and dissection of different organs
Five male rats (7 weeks old, 240-260g for each) were used for this study. The
rats were anaesthetized by intraperitoneal injection of a mixture of Narcotic solution
(use 0.1ml for 10g bodyweight, Narcotic solution recipe see Table 3). Then the rats
were soon dissected and organs (brain, heart, kidney, liver, lung, muscle, pituitary,
spleen, testis) were kept in liquid nitrogen for further total RNA isolation.
3.6.2 Tissue total RNA isolation
Before isolation of total RNA, the bench surface, glassware and plastic ware
were cleaned with RNase AWAY®. The rat samples frozen in liquid nitrogen were
melted on ice. 60mg of tissue should be quickly cut into slices with less than 0.5cm
thickness and submerged into 600µl RLT Buffer (containing 1% β-mercaptoethanol and
3% Proteinase K) supplied in the Qiagen RNeasy® Mini Kit. The dispergierer IKA
Ultra-Turrax® T25 was used to disperse and disrupt the cells from the tissues. Whenever
another tissue sample was operated, the dispergierer needed to be rinsed with SDS in
DEPC water for 10 seconds and with 70% ethanol in DEPC water for 10 seconds.
Thereafter, the tissues were homogenized using the clean dispergierer IKA UltraTurrax® T25, and the cell debris was spun down by centrifugation at 10,000×g for
3min. The supernatant was transferred into a new tube and 0.5 volumes of pure ethanol
were added and mixed gently. The mixture from fiber-rich tissues (such as heat and
muscle) was handeled according to the Qiagen RNeasy® Fibrous Tissue Mini Kit
protocol①, and the other mixtures from easy-to-lyse tissues were handeled according to
the Qiagen RNeasy® Mini Kit protocol②. The total RNA was stored in RNase-free water
at -80°C and the RNA quality was examined before further use. In this study, the RNA
quality was checked by running a RNA-Chip with a Bioanalyzer (Figure 15 and
Supplemental data S2).
①
https://www.qiagen.com/de/resources/resourcedetail?id=8039840d-4815-4375-8933bc09247e47c0&lang=en
②
https://www.qiagen.com/de/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbcbf9f6fa33e24&lang=en
Page | 59
Figure 15 An example of RNA-Chip results to check the quality of isolated RNAs.
3.6.3 Ethanol precipitation to concentrate total RNA
50µl of total RNA sample needed to be mixed with 125µl of 100% ethanol and
5µl sodium citrate (pH 5.2) and incubated at -20°C for one hour. Thereafter, the mixture
was centrifuged at 13,000rpm for 30 minutes at 4°C. The supernatant was discarded and
the pellet was resuspended in 500µl of 70% ethanol for washing. Thereafter, the
centrifugation was repeated under the same condition and the supernatant was
discarded. The final RNA pellet was solved in RNase free water and kept in -80°C.
3.7 Workflow for Northern blotting
Northern blotting is a method to detect target mRNA by specific RNA probes.
We planed to eventually reveal distinct alternatively spliced forms of Akap11 transcripts
([U48288.1] 9.7k bases and [NM_012773.2] 7k bases, Figure 29) by a non-radioactive
method. Figure 16 depicts a work flow of the probe generation, Northern blotting
hybridization and detection steps. The RNA probe generation and validation is
described in details in the following subsection 3.7.1 and 3.7.2.
Page | 60
Analysis of target gene and
specific primers design
PCR amplification of target
gene and housekeeping gene
Ligate purified PCR product into
pGEM-T vector by TA-cloning
Competent E.coli transformation,
colony screening, insert fragment
validation
Restriction enzyme site analysis and
plasmid linearization
DIG-labelled RNA
probe generation
RNA isolation
RNA separation
by agarose gel
electrophoresis
Transfer RNA to positive
charged nylon membrane
RNA fixation
by heat and UV
Membrane hybridized with
DIG-labelled RNA probes
Visualization of fixed RNA
on nylon membrane by
methylene blue
Weight
Paper tower
pre-wet blotting paper
positive charged nylon membrane
Agarose gel
slim parafilm
pre-wet thick blotting paper
A glass support
10×SSC Transfer buffer
Visualization of labelled
RNA on film by
immunodetection
Figure 16 workflow of Northern blot
Page | 61
3.7.1 Plasmid construction and probe generation by in vitro
RNA transcription
The AKAP11 clone3 plasmid was bought from GenomeCube. The plasmid was
sequenced from both SP6 and T7 promotors until fully overlapping. According to the
sequence feature analysis of the AKAP11 clone3 plasmid, a fragment of 448bp was
suitable to design primers for a rat AKAP11 mRNA [NM_012773.2] specific probe. A
fragment of 2615bp was suitable to design probes for the two rat AKAP11 mRNAs both
[NM_012773.2] and [U48288.1], available in the NCBI database. As controls,
sequences for 28S (Lab-ID: G12/14), GAPDH (Lab-ID: E12/15), β-actin (Lab-ID:
M29/30), StAR (Lab-ID: K21/22) probes were amplified with specific primers. All
specific PCR products were cloned by TA-cloning into the pGEM-Teasy vector.
Positive clones were identified by colony PCR and subsequent plasmid sequencing. All
plasmids were sequenced to identify the orientation and precise length of the insert
sequences. Thereafter, it was decided which promoter (SP6 or T7) had to be used for
antisense RNA-probe in vitro transcription. Linearized plasmids were used to generate
RNA probes according to the instruction of the “DIG RNA Labeling Mix, Roche”①.
Quality and quantity of the transcripts were analyzed by non-denaturing agarose gel
electrophoresis and ethidium bromide staining. According to the kit description, the
signal from the in vitro transcribed RNA band should be about 10 fold stronger than that
from the DNA used for amplification.
3.7.2 Dot blot assay for detection of DIG-labeling efficiency
A dot blot assay was used to determine the labeling efficiency and sensitivity of
the DIG-labeling reaction. Dilutions of 100ng/µl, 10ng/µl, 2ng/µl, 500pg/µl, 100pg/µl,
50pg/µl, 25pg/µl, 10pg/µl, 5pg/µl, 2.5pg/µl, 1pg/µl of the DIG-labelled RNA probes in
sterile RNase-free ddH2O, were spotted in a drop of 1µl for each dilution onto a
positive-charged nylon membrane. After RNA fixation by 30min baking at 80°C and
30s UV-treatment at 1.5J/cm2, staining of the RNA on the membrane was done in
0.03% (w/v) methylene blue in 0.3M sodium acetate (pH 5.2) for 1min, followed by
destaining in DEPC water for 2min. The immunological detection of the labeled probe
was performed with a chemiluminescence procedure. Ideally, DIG-probes exhibited a
detectable spot in a concentration as low as 0.1pg/µl. Probes with a detectability of at
①
http://netdocs.roche.com/DDM/Effective/pdf_0900b2fc80acecf6.pdf
Page | 62
least 1pg/µl are required to obtain positive signal on Northern blots. The protocol was
modified in overall steps from the Roche manufacture’s instruction and Box3in
Zimmerman’s Nature publication (Zimmerman et al., 2013).
3.7.3 RNA samples and agarose gel preparation
The denaturing agarose gel used for RNA electrophoresis was made by 0.1%
DEPC water, containing 1.8% agarose, 1×MOPS, 6% Formaldehyde and 0.4µg/ml
Ethidium bromide. 1-10µg RNA was solved in DEPC water, containing 1×MOPS, 6%
Formaldehyde, 42% Formamide and 1× loading buffer. RNA samples were denatured
by heating at 65°C for 15min. The electrophoresis was done in 1×MOPS running buffer
at 20V overnight.
3.7.4 Protocol of Northern blotting
1-10µg total RNA sample (Table 17 and 18) in high quality (no degradation,
“pure” RNA: A260/280 = ~2.0, A260/230 = 2.0~2.2 (Scientific, 2013)) was denatured and
loaded to a 1% agarose gel and separated by gel electrophoresis at 20V overnight
(17h30min). The RNA was transferred by capillary blotting (blotting “sandwich”,
Figure 16) from the gel to a positive-charged nylon membrane. The following Northern
blotting procedure is a modified form of the instruction of the “DIG Northern Starter
Kit” and Nature Protocols (Streit et al., 2008; Zimmerman et al., 2013).
The nylon membrane with the total RNA on top was rinsed with 6×SSC buffer
for 5min followed by air drying. Thereafter, the membrane was baked at 80°C in an
oven for 30min (RNA-side-up). Additional RNA fixation was done by cross-linking
was in a UV-trans-illuminator at 1.5J/cm2 for 30s (RNA-side-up). The membrane with
fixed RNA was rinsed with ddH2O again and stained with Methylene Blue for 1min to
indicate the position of the RNA marker, 18S and 28S rRNA and reveal the transfered
total RNA. The blue color could be washed away by ddH2O. To be prepared for the
RNA hybridization, the membrane should be first rinsed with 6×SSC buffer and then
incubated in a formamide containing prehybridization buffer (1ml/10cm2 membrane) in
a glass tube for 3h with rotation in a hybridization oven at a constant temperature of
42°C. The DIG-labeled specific RNA probe was denatured at 65°C for 15min before
use and kept on ice for 10min. The membrane with total RNAs was incubated with the
denatured RNA probe at the concentration of 100ng probe/ml hybridization buffer and
continuously rotated at 42°C in the hybridization oven overnight. The next morning, the
Page | 63
hybridization buffer was discarded and the same amount of 2×SSC was added for 5min
rotation at room temperature. Thereafter, the membrane was rinsed by 1×SSC for
10min and incubated with Roche protein blocking buffer for 40min. For the 1st antibody
incubation, mouse anti-DIG monoclonal unconjugated antibody was used at the
working dilution of 3: 5000 in blocking buffer for 30min at RT. The membrane was
washed with washing buffer 1×SSC for four times 10min each at RT. The 2nd antibody
anti-mouse IgG was used at a working dilution of 1: 30,000 in washing buffer for 45min
incubation. Thereafter, the membrane was washed five times with 1×SSC washing
buffer for 10min each to remove the unspecific binding. Immune-Star™ AP was used as
substrate to detect the hybridization signal in the dark room.
The stripping protocol from Roche suggested to rinse the membrane thoroughly
in DEPC water and invubate it at 80°C in stripping buffer for 2× 60min, followed
another 5min rinse thoroughly in 2×SSC buffer. However this stripping protocol proved
to be not optimal in this study, wherefore this is not recommended and a new one
needed to be developed.
3.8 Construction of myc-tagged AKAP plasmids and expression in
TM4 cells
By the bioinfomatic screening for the AKAPs with putative PTS1 signals, we
were able to find three potential conserved “peroxisomal” AKAP subfamilies (AKAP1,
AKAP4 and AKAP10) in the first round rough screening (Figure 17). Eventhough the
tripeptide “SKL”-variant was contained in all species analyzed, the PTS1 finder used in
in the second round PTS1 screening did recognize these signals as putative peroxisomal
targeting signal 1, but not jadged them as peroxisomal AKAPs (Figure 38-40).
Subcellular localization of a protein should, however, not only rely on computer
predictions, but also revealed by experimental comfirmation. Since our group had
already bought many commercially available antibodies against several peroxisomal
proteins which showed various cross-reactions with other non-specific proteins in
homogenates, we decided to clone several interesting AKAP-families members with
putative “SKL”-variants. Therefore, AKAPs with different myc tags were constructed
and myc-tagged GFP was used as control for the protein overexpression in Sertoli cells.
For each AKAP and the GFP control, two types of recombinants were used for the
Page | 64
expression analysis and subcellular localization: myc tag at the amino-terminus & myc
tag at the carboxyl-terminus of the fusion protein.
3.8.1 Construction of myc-tagged AKAPs and GFP plasmids
Three IMAGE clones (Table 4 pYX-Asc-AKAP1, pCR-Blunt II-TOPO-AKAP4,
and pSPORT 1-AKAP10) were selected and purchased from GenomeCube as the
templates for whole length amplification of AKAP1, 4 and 10. At the same time, the
pGeneClip™ hMGFP vector was used as the template for the recombinant positive
control. Thereafter, for two types vectors expression primers were designed (Table 5).
Two Taq-PCRs were performed (Table 8) to amplify whole GFP cDNA (684bp) and
Six Pfu-PCRs were performed (Table 9) to amplify accurately the AKAP complete
cDNAs (Akap1 cDNA 2589bp; Akap4 cDNA 2565bp; Akap10 cDNA 1131bp).
Table 8 Taq-PCR reaction
GFP_3B(BL48/BE66); GFP_5B(BE65/BE67)
pGeneClip™ hMGFP Vector [AY744386.1]
Reagent
Volume
Taq PCR 10× Buffer
2.5µl
dNTP mix (10mM each)
0.5µl
primer mix (10µM)
2µl
Taq DNA polymerase (5U/µl)
0.5µl
plasmid (5ng/µl)
2µl
LONZA H2O
18µl
25µl
Taq-PCR procedure: 95°C for 2min initiate denaturation, followed by 35 cycles
of denaturation at 95°C for 30s, annealing at 58°C for 30s, elongation at 72°C for
5min30sand one final extension step at 72°C for 5min.
Table 9 Pfu-PCR reaction
Reagent
Volume
10× Pfu Buffer
2.5µl
0.5µl
Page | 65
AKAP1_3B(BH42/BH43); AKAP1_5B(BH48/BH49); AKAP4_3B(BH44/BH45);
primer mix (10µM)
2µl
Pfu DNA
0.5µl
AKAP4_5B(BH50/BH51); AKAP10_3B(BL28/BH47); AKAP10_5B(BL29/BH53)
polymerase(2U/µl)
AKAP1_IMAGE [BC065135.1]; AKAP4_IMAGE [BC130271.1];
plasmid (5ng/µl)
2µl
LONZA H2O
18µl
AKAP10_IMAGE [BC054105.1]
25µl
The Pfu-PCR procedure was as follows: initial denaturation at 95°C for 2min,
followed by 35 cycles of 95°C for 30s denaturation, 58°C for 30s annealing, 72°C for
5min30s elongation and a final extention step at 72°C for 5min.
Compared to the Taq DNA polymerase, at one hand, Pfu polymerase possesses a
higher thermostability and fewer false proofreading properties, because Pfu is able to
correct errors by its 3’-5’ exonuclease activity during DNA amplification. At the other
hand, DNA products made by Pfu are not useful in direct TA cloning, since they are
lacking of “A (adenine)” 3’overhangs at their blunt ends. Therefore, blunt end PCR
fragment ligation needs an extra “dATP” addtion to the blunt end.
All the Pfu-PCR products were therefore purified by regular gel extraction
method. 7µl of the eluted PCR fragment was mixed with 1µl Taq PCR 10× Buffer
(25mM MgCl2), 1µl 2mM dATP, 5 units of Taq DNA polymerase, and deionized H2O
(LONZA H2O) to a final volume of 10µl. Thereafter, this 10µl mixture was incubated at
70°C for 30min to add an extra “dATP” to the blunt end. This procedure for blunt end
PCR fragment ligation was slightly modified from Promega protocol①.
Following the regular TA cloning procedure, a 12µl ligation reaction including
5µl of the PCR fragment mixture, 1µl pGEM-T easy vector, 5µl 2× ligation buffer and
1µl T4 ligase was incubated at 4°C overnight (14-16 hours).
The two types of GFP ligation were transformed into E.coli (see subsection
3.8.2) and sent to verify the sequences. Thereafter, the two types of GFP were
subcloned into the expression vector pCMV-3B and pCMV-5B, respectively (Table 10
①
http://www.promega.com/~/media/files/resources/promega%20notes/62/cloning%20bl
untend%20dna%20fragments%20into%20the%20pgem-t%20vector%20systems.pdf
Page | 66
and Table 11). These two types recombinants were sequenced to make sure that GFP
was in-frame with the myc-tag. The transfection of two types myc-tagged GFP were to
double assess the cytoplasmic localization of GFP by its green fluorescence and the
myc-tag by indiect red fluorescence with a mouse anti-myc secondary antibody
(subsection 3.8.4-3.8.6). With this procedure, a perfect colocalization of GFP and the
myc-tag was observed, suggesting that the used anti-myc antibody was excellent and
suitable to use for the detections of the myc-tagged AKAP constructs.
The procedure for the three AKAP-recombinant plasmid constructions was
following similar protocols as ones for the GFP contructs. Only AKAP1 needed an
additional point mutation assay before being ligated into the expression vectors
(subsection 3.8.3) because of an unexpected stop codon mutation.
Table 10 Restriction enzyme digestion of GFP and 3 AKAPs (4h incubation at 37°C)
Reagent
Volume Reagent
Volume Reagent
Volume
LONZA H2O
Xµl
LONZA H2O
Xµl
LONZA H2O
Xµl
AKAP1-pGEM-T
(total 3.75µg)
Yµl
AKAP4-pGEM-T
(total 3.75µg)
Yµl
AKAP10-pGEM-T
(total 3.75µg)
Yµl
RE 10× Buffer B
5µl
RE 10× Buffer B
5µl
RE 10× Buffer D
5µl
Acetylated BSA
0.5µl
Acetylated BSA
0.5µl
Acetylated BSA
0.5µl
BamHI (10U/µl)
1.25µl
BamHI (10U/µl)
1.25µl
EcoRI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
50µl
50µl
50µl
Reagent
Volume
Reagent
Volume
Reagent
Volume
LONZA H2O
Xµl
LONZA H2O
Xµl
LONZA H2O
Xµl
GFP-pGEM-T (total
3.75µg)
Yµl
pCMV-3B or pCMV5B (total 3.75µg)
Yµl
pCMV-3B or pCMV5B (total 3.75µg)
Yµl
RE 10× Buffer B
5µl
RE 10× Buffer B
5µl
RE 10× Buffer D
5µl
Acetylated BSA
0.5µl
Acetylated BSA
0.5µl
Acetylated BSA
0.5µl
BamHI (10U/µl)
1.25µl
BamHI (10U/µl)
1.25µl
EcoRI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
XhoI (10U/µl)
1.25µl
50µl
50µl
50µl
Page | 67
Table 11 Ligation reaction (overnight incubation at 4°C)
Reagent
Volume
Digested and purified insert fragment (total 500ng)
Xµl
Digested and purified vector fragment (total 200ng)
Yµl
10× ligation Buffer
2µl
T4 ligase
1µl
20µl
3.8.2 Procedures for bacterial transformation
3.8.2.1 Heat shock procedure
The LB/Ampicillinr (100µg/ml) plates were prepared freshly and store in the
clean bench prior to use. 5µl ligation reaction were added into 50µl DH5α competent
cells aliquot and mixed gently by pipetting. After 20 minutes incubation on ice, a 90
seconds heat-shock was performed in a water bath with a constant temperature of 42°C.
The shocked cells were immediately chilled on ice for 2 minutes. 200µl of SOC
medium were added to grow the transformed cells at 37°C for 30 minutes incubation.
During the incubation period, a mixture of 100µl LB, 20µl IPTG (1M) and 40µl X-gal
(2%) was spreaded onto the Ampr plates, followed by air drying. The complete 255µl
transformation culture of the tube was spread onto the Ampr plate. A plasmid as the
positive control had been previous transformed efficiently and an Ampr plate with
DH5α alone was used as the negative control. After an overnight incubation at 37°C, the
blue white screening was used to identify successfully ligated clone. This protocol was
slightly modified from the original protocol of promega①.
3.8.2.2 Electroporation procedure
Efficient introduction of DNA into bacteria is critical for the success of many
molecular biology procedures. The electroporation method for transforming E.coli cells
①
https://www.promega.com/~/media/files/resources/protcards/pgem%20t%20and%20pg
em%20t%20easy%20vector%20systems%20quick%20protocol.pdf
Page | 68
can produce efficiencies greater than those achieved with the best chemical methods①,
especially for large plasmids (4.1kb-6.9kb). Commericial avialable ElectroTen-Blue®
cells needed only to be thawed and mixed with DNA prior to electroporation.
The genotype of bacteria DH5α used for heat shock transformation is F–
Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44
λ– thi-1 gyrA96 relA1 ② . The DH5α exhibits no antibiotic resistance. However, some
electroporation competent cells possess resistance to antibiotics.
The electroporation competent cell used in our lab is called “EletroTen-Blue®”.
It is a derivative of the XL1-Blue MRF´strain that can withstand much higher levels of
electrical current. This increases the survival of the cells during electroporation, and
thus the efficiency of the ligated constructs transformation. In addition, EletroTen-Blue
strain genotype is Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1
gyrA96 relA1 lac Kanr [F´proAB lacIq ZΔM15 Tn10 (Tetr )], which are resistant to
both kanamycin and tetracycline. Thus plasmids that carry the Kanamycin-resistance or
Tetracycline-resistance selection marker cannot be propagated in this strain③.
Moreover, from the structural point of view, Neomycin whose chemical
structure is similar to Kanamycin (Table 12) can also not be used for selection of
transformed EletroTen-Blue clones. Therefore, the expression plasmids pCMV-Tag 3B
and pCMV-Tag 5B could not be used for this electroporation procedure, since they have
Neor/Kanr selection, while the clonning vector pGEM-T, ideal for Ampr selection, was
suitable for EletroTen-Blue cells.
①
http://www.chem.agilent.com/library/usermanuals/Public/200159.pdf
http://www.lifetechnologies.com/de/de/home/life-science/cloning/competent-cells-fortransformation/chemically-competent/dh5alpha-genotypes.html
③
http://www.chem.agilent.com/library/usermanuals/Public/200159.pdf
②
Page | 69
Table 12 Chemical structure of commen used antibiotics
Antibiotics
Tetracycline
Kanamycin
Neomycin
Ampicillin
Structure
Antibiotics
Structure
3.8.3 Point mutation assay
Aligned with AKAP1 mRNA variant 2 [NM_001042541.1], the sequenced
AKAP1_IMAGE
clone
cDNA
[BC065135.1]
contained
2
mutation
points:
TAA(STOP)1993GAA(Glu) and TCT(Ser)2208TCC(Ser). The second point mutation
(C2208T) caused no amino acid change, which could be neglected. But the first point
mutation (G1993T) caused an extra STOP codon, which had to be mutated back to the
original base pairs.
Table 13 AKAP1-Mutation-PCR reagent
Reagent
Volume
10× Pfu Buffer
5µl
0.8µl
AKAP1_Mut_F (10pmol/µl) / AKAP1_Mut_R (10pmol/µl)
primer mix (10µM)
2µl
pGEM-T-AKAP1_3B; pGEM-T-AKAP1_5B
plasmid (20ng/µl)
1µl
Pfu DNA polymerase
1µl
DMSO
2.5µl
LONZA H2O
38µl
50µl
Page | 70
The Mut-PCR procedure was as follows: initial denaturation at 94°C for
1min30s followed by 18 cycles of 94°C for 30s denaturation, 55°C for 1min annealing,
68°C for 13min elongation and a final extention step at 68°C for 13min.
1 tube of PCR reagent without Mut-Primers was set as the negative control. The
DNA template was digested completely by DpnI and no colony were observed on Ampr
plate. Unmutated plasmids had been previous transformed efficiently to DH5α as
positive control to verify the transformation procedure.
Table 14 AKAP1-Mutation-PCR Dpn I digestion (4h incubation at 37°C)
Reagent
Volume
pGEM-T-AKAP1-3B_Mut; pGEM-T-AKAP1-5B_Mut
Purified mutation plasmid
42µl
10× Buffer B
5µl
BSA (10mg/µl)
0.5µl
DpnI
2.5µl
50µl
15µl digested plasmids (samples or negative control) or 5µl ligated plasmids
(positive control) were transformed to DH5α by the heat shock procedure (see
subsection 3.8.2.1). Clones with the correct mutated sequence verified by sequencing
were subcloned into expression vectors pCMV-3B and pCMV-5B (Table 10 and Table
11).
3.8.4 Coating cover slips with poly-L-lysine
10mg of poly-L-lysine was dissolved in 100ml H3BO3/Na-tetraborat buffer (see
Table 3 Buffer recipes). The solution needed to be sterilized through 0.22µm Millipore
filtration. Moreover, 12mm cover slips were sterilized by 4h baking at 180°C. All the
glassware and materials used were sterile and all operations performed in a laminar
hood. The cover slips were incubated in poly-L-lysine solution in a 3.5 mm Petri dish
overnight. On the next morning, the poly-L-lysine solution was removed by aspiration
and the cover slips were incubated with sterile water in Petri dish for 5h at RT.
Thereafter, the coated cover slips were rinsed with sterile water twice and air dried in
the hood prior to storage at 4°C.
Page | 71
3.8.5 Transient transfection of TM4 cells
One day before transfection, the TM4 cells need to be sown on the poly-L-lysine
coated coverslips at a density of 5×104 cell/well with 500µl serum containing medium
in a 24-well plate. After 24h growth (37°C, 5%CO2), cells were approximately 75%
confluent. Transient transfection was then carried out according to the Promega protocol
by using the ViaFect™ Transfection Reagent ① . For this study, all the plasmids for
transfection were purified with an endotoxin-free plasmid DNA purification kit
(NucleoBond® Xtra Midi EF, Macherey-Nagel Co.). 500ng plasmid DNA (myc-GFP
plasmids used as positive control) and 3µl reagent were mixed with 500µl serum free
medium per well. The transfection efficiency on TM4 cells was about 15% after oneday growth. On the second day of transfection, the cells were ready for
immunofluorescence staining.
3.8.6 Immunofluorescence protocol
TM4 cells, grown on poly-L-lysine coated coverslips for 48h, were fixed with
4% paraformaldehyde in PBS for 30min at room temperature. After 3 times washing
with warm PBS for 10min each, cells were treated with 1% glycin or/and 0.1% Triton
X-100 for permeablization. Cells were washed 3 times with PBS for 5min each, and
blocked for 1h in 1% PBSA with 0.05% Tween, thereafter incubated with primary
antibody (dilutions described in Table 6) overnight at room temperature. The next day,
cells were washed 3 times with PBS for 5min each, followed by 1h secondary antibody
incubation in the dark. Thereafter, cells were washed with PBS for 10min, followed by
nuclear staining and final washing of 2×10min. The coverslips were mounted with the
cell-side down onto glass slides with Mowiol 4-88 containing propylgallat mixture as
anti-bleaching agent. The staining pattern was checked and pictures were taken with a
fluorescence microscope (Leica DMR) or a laserscan confocal microscope (Leica TCS
SP2) using 63× oil immersion objectives.
①
https://www.promega.de/~/media/files/resources/protcards/viafect%20transfection%20
reagent%20quick%20protocol.pdf
Page | 72
4 Results
Follicle-stimulating hormone (FSH)-mediated flux through metabolic pathways
is controlled by secondary modification of proteins such as phosphorylation and
dephosphorylation at specific subcellular locations. This regulation has been shown for
mitochondrial cholesterol influx to occur via the AKAP1-StAR interaction. However,
very limited knowledge is available to date on the direct control of these metabolic
pathways in peroxisomes.
4.1 AKAP family members with putative PTS1 signals
Since a previous report showed that AKAP11 is probably localized to the
peroxisome, it is interesting to determine whether other proteins of AKAP family are
putative peroxisomal proteins. Therefore, I screened for AKAP isoforms with putative
peroxisomal targeting signals.
4.1.1 AKAP sequence retrieval and PTS1 search matrix
All known AKAP sequences (1306 sequences) from diverse species were
retrieved from “UniProt” database① followed by the screening for a putative C-terminal
PTS1. Only the C-terminal tripeptide was used to screen for the putative C-terminal
PTS1, since it is the crucial portion for peroxisome import. After removing the
duplicates, 531 different tripeptides were left over from all AKAP sequences
(Supplemental data S3).
For classification of all variants of possible PTS1 signals, all known PTS1
binding sites from the “Peroxisomal Target Signal Predictor (PTSP)” were downloaded
(Supplemental data S4) and summarized into a Combination Matrix (Table 15).
①
http://www.uniprot.org/uniprot/
Page | 73
Table 15 Summarized PTS1 tripeptides derived from the “Peroxisomal Target Signal Predictor (PTSP)”
PTS1 tripeptides in standard amino acid 1-letter code
A
A
F
AHL
CKL
PRL
SNL
C
H
I
AKF
CRL
QAL
SQL
H
K
L
AKI
HKL
SHL
SRI
K
N
M
AKL
HRL
SKI
SRL
N
Q
V
P
R
Q
S
ANL
KKL
SKL
SRM
ARL
NKL
SKM
SSL
ASL
PKL
SKV
THL
S
T
4.1.2 Phylogenetic analysis of AKAP proteins containing a
putative PTS1 signal
Using the combination matrix for conserved PTS1 substitutions, 81 AKAP
proteins with possible PTS1 ends were identified and used to generate a phylogenetic
tree (Figure 17). The AKAPs clustered into six large groups belonging to the AKAP1,
3, 4, 10, 11, 14 and 17A families, while two sequences distributed into distinct groups
as individual entities. An unexpected finding was that the short form of AKAP11 from
the rat (sp|Q62924|AKAP11 Rat-CRL) clustered into a separate group. Interestingly,
AKAP family members within the described groups displayed similar PTS1 signals,
suggesting that the signal was conserved during evolution and that they might be real
peroxisomal targeting signals.
Page | 74
AKAP4
AKAP3
AKAP11
AKAP17A
AKAP10
AKAP14
AKAP1
Figure 17 Phylogenetic tree of AKAPs with putative PTS1
Page | 75
By analysis of the EST expression profile I found that the AKAP1, 4, 10
families are highly expressed in testis (Figure 41-42). The sequence and the
phylogenetic analysis of these AKAPs indicated that they might contain a putative
PTS1 signal, however, these proteins were reported to be localized to the mitochondria
or other subcellular sites and not the peroxisome (see subsection 1.4.2-1.4.4). However,
peroxisomal localization of proteins has been overlooked quite frequently in the past
and some peroxisomal protein isoforms have multiple subcellular locations (Islinger et
al., 2009). Therefore, it was speculated that depending on the cellular requirements and
developmental stages, AKAPs might be localized in different subcellular compartments
(e.g. mitochondria and peroxisome). In section 4.6, the subcellular location of myc
epitope tagged AKAP1, AKAP4 and AKAP10 in TM4 Sertoli cell line were analyzed.
4.2 TM4 Sertoli cell culture stimulation with FSH treatment
To study the regulation of the peroxisomal compartment by FSH signaling and
AKAPs, a good cell culture model was established.
4.2.1 Cell culture condition optimization
Follicle-stimulating hormone (FSH) binds to the its receptor on the plasma
membrane of Sertoli cells of the testis and granulosa cells of the ovary, thereby
initiating an intracellular signaling cascade inducing the activation of PKA to
phosphorylate downstream target proteins, which are essential for the regulation of
androgen and progesterone production and sperm motility (Tasken and Aandahl, 2004;
Walker and Cheng, 2005). It is known that peroxisomes play an important role in
spermatogenesis and oxidative stress protection in the testis (Dastig et al., 2011) and
that Sertoli cells in the testis contain high levels of metabolic peroxisomal proteins
(Nenicu et al., 2007). However, it was not known whether peroxisomes or peroxisomal
genes are affected by activation of the FSH signaling pathway. To study potential effect
of FSH on the peroxisomal compartment, the mouse Sertoli cell line-TM4 cells were
purchased from the ATCC. The exact cell culture condition of the TM4 Sertoli cells was
established by seeding them at different densities (Table 16)
Page | 76
Table 16 Optimization of cell density to obtain a monolayer of TM4 cells on Day 4-6
The 1st set
Density of cells
Day 4
Day 6
seeded per ml
A Group
2 × 10
No cells
Almost no cells, all cells are round
B Group
2 × 102
Very few cells
Still no connection, too many round
cells
C Group
2 × 103
No cluster, no connection
Some part is monolayer, some part
no connection
D Group
2 × 104
Some part is monolayer,
some part is multilayer
Too full
E Group
2 × 105
Multilayer, overgrown
Too full
F Group
2 × 106
Too full
Too full
The 2nd set
Density of cells
seeded per ml
Day 4
Day 5
G Group
3 × 103
No connection, single cell
Most part no connection, some part
is monolayer
H Group
6 × 103
Some part is monolayer,
some part is single cell
Most part is monolayer, some part no
connection
I Group
1.2 × 104
Monolayer, perfect!
Half part is monolayer, half part is
multilayer
The best monolayer at Day 4 was obtained with a seeding density of 2.34×103
cells/cm2 = 9 × 103 cells/ml. This experimental condition was used further for
stimulation of TM4 Sertoli cells with FSH stimulation.
4.2.2 Effect of FSH stimulation on the peroxisome gene
expression
After optimization of the culturing conditions, TM4 cells were treated with
increasing FSH concentration (W0 = 0ng/ml, W1 = 10ng/ml, W2 = 100ng/ml, W3 =
1000ng/ml). From all four experimental groups (W0, W1, W2, W3), total RNA was
isolated, reverse transcribed and used for semiquantitative analysis of mRNA
expression of genes involved in peroxisomal biogenesis and proliferation, antioxidative
enzymes and lipid metabolism (Figure 18-24).
Page | 77
fold induction of mRNA level
2,5
2,0
W0
1,5
W1
1,0
W2
W3
0,5
0,0
28S
rRNA
GAPDH
FSHR
StAR
Figure 18 Measurement of the mRNA of internal control and positive controls by conventional RT-PCR
FSH receptor is present in TM4 cells. The FSH stimulation of TM4 cells worked
well with 1000ng/ml, since Star gene was upregulated, whereas GAPDH and FSHR
were not altered (Figure 18). The calculations for Figure 18-24 were made with the 28S
rRNA values from Figure 18.
3,0
2,5
2,0
W0
1,5
W1
1,0
W2
0,5
W3
0,0
28S
rRNA
Pex11
alpha
Pex11 Pex11
beta gamma
Figure 19 Alterations of mRNA levels of peroxins on peroxisome proliferation
There is a clear indication for the Pex11α, the inducible form of the PEX11
proteins, whereas the other family members were not changed (Figure 19).
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
W0
W1
W2
W3
28S
rRNA
Pex5
Pex13
Figure 20 Alterations of mRNA levels of peroxins on peroxisomal matrix protein import
Page | 78
Pex14
In comparison to the Pex11α gene, some maker genes for peroxisomal matrix
protein import (Pex5, Pex14) were not altered while Pex13 showed a slight upregulation
in gel measurements (Figure 20).
2,5
2,0
W0
1,5
W1
1,0
W2
0,5
W3
0,0
28S Catalase GPX1
rRNA
SOD1
Figure 21 Quantification of alterations of mRNA levels of antioxidative enzymes by conventional RT-PCR
FSH treatment leads to the upregulation of the catalase expression, whereas
Gpx1 and Sod1 were not altered. This is of interest, since catalase is the only one
protein involved in anti-oxidation metabolism that is exclusively present in
peroxisomes, whereas Gpx1 and Sod1 are also present in other cellular compartments
(Figure 21).
2,0
1,8
1,6
1,4
1,2
W0
1,0
W1
0,8
W2
0,6
W3
0,4
0,2
0,0
28S
rRNA
ACOX1 ACOX2 ACOX3
Figure 22 Alterations of mRNA levels of Acyl-CoA oxidases by FSH treatment
From the three Acyl-CoA oxidase mRNAs, only the one of Acox2 showed a
significant upregulation (Figure 22). This is of interest, since this enzyme is involved in
the conversion of the cholesterol side-chain, whereas the other members of the Acox
Page | 79
family degrade mainly straight chain- and branched chain fatty acids (Baumgart et al.,
fold of induction mRNA level
1996b).
1,6
1,4
1,2
1,0
W0
0,8
W1
0,6
W2
0,4
W3
0,2
0,0
28S
rRNA
ABCD1 ABCD2 ABCD3
Figure 23 Conventional RT-PCR results of alterations of mRNA levels of fatty acid transporters
ABCD1-ABCD3 are the members of ATP-binding cassette (ABC) transporters
superfamily in peroxisomal membrane and play a role in transporting fatty acids for the
lipid metabolism into the peroxisomal matrix. From the fatty acid transporters ABCD1ABCD3, mainly ABCD2 is upregulated by the increasing FSH stimulation. These 3
members of ABCD proteins were found to have overlapping substrates as well as
distinct specificities (van Roermund et al., 2014). ABCD1 (also called ALDP)
preferentially transported saturated VLCFA such as C24:0 and C26:0 as substrates for
β-oxidation activity, and the X-ALD patients (X-linked adrenoleukodystrophy) with
genetic defective ABCD1 exhibit the accumulation of VLCFA in plasma. The
substrates of ABCD2 (also called ALDRP) were C22:0 and also unsaturated VLCFA
such as C24:6 and C22:6 (van Roermund et al., 2011). ABCD3 (also called PMP70)
was suggested to transport long branched chain fatty acids, long-chain unsaturated- and
dicarboxylic fatty acids (C27 bile acids) across the peroxisomal membrane, which is
important for bile acid biosynthesis (Ferdinandusse et al., 2014; van Roermund et al.,
2014).
Figure 24 Measurement of PPAR
transcription factors after treatment
with different concentration of FSH
Page | 80
Since many peroxisomal genes are regulated by PPARs, PPARα, -β, -γ
expression in TM4 Sertoli cells were also analyzed. Indeed, the mRNAs of all PPAR
family members were expressed in Sertoli cell line, with the PPARβ mRNA showing
the strongest signal (Figure 24). PPARα performs multiple functions in the liver, heart
and blood vessel wall, and is also known to be activated by FSH to compose
phospholipids in germ cells. PPARβ is present ubiquitously and plays a role in glucose
homeostasis and fatty acid oxidation. PPARγ is mainly expressed in mature Sertoli
cells, adipocytes and skeletal muscles and takes part in anti-diabetic and antiatherosclerotic mechanisms (Kota et al., 2005; Thomas et al., 2010). PPARs are also
known to be involved in the regulation of fertility. However, there was no major change
in their mRNA levels after FSH treatment.
4.3 Isolation of peroxisomes from TM4 cells
AKAP220, the short form of AKAP11 in the rat (a single frame in the Figure
17), was the only AKAP protein to be reported in peroxisomes. I tried to repeat and
establish the peroxisomal localization in our laboratory to study effects of FSHstimulation of this protein on the peroxisomal compartment. However, this protein
clustered singly into a separate group from all other AKAP family members in the
phylogenetic tree. It was speculated that there might be a problem with the peroxisomal
localization of this protein reported by Scott group (Lester et al., 1996). Therefore,
attempt was made to localize this protein from highly purified peroxisomal fractions by
antibody-based test. To localize AKAP11 in peroxisomes, the differential centrifugation
and OptiPrep density gradient centrifugation were used to isolate pure peroxisomal
fractions followed by detection with a commercially available anti-AKAP220 antibody.
Page | 81
Figure 25 Peroxisome marker PEX14p (60 kDa) distribution. H1: homogenization; S1: supernatant after
1st centrifugation from H1; P1: pellet after 1st centrifugation from H1, mainly contains cell debris; S2:
supernatant after 2nd centrifugation from S1; P2: pellet after 2 nd centrifugation from S1, mainly contains
heavy mitochondria and some peroxisomes; S3: supernatant after 3rd centrifugation from S2; P3: pellet
after 3rd centrifugation from S2, mainly contains light mitochondria and enriched peroxisome, using for
the further density gradient centrifugation; S4: supernatant after 4 th centrifugation from S3, mainly
contains cytoplasm substance; P4: pellet after 4th centrifugation from S3, mainly contains microsome and
small peroxisome. Then P3 was taken for 2h30min OptiPrep density gradient centrifugation. B1 to B12
are the 12 tubes fraction of handled OptiPrep through constant speed pump.
Figure 26 Western blot of AKAP220 (AKAP220 should be 220 kDa). The samples H1to P4 and B1 to
B12 are the same as Figure 25.
Page | 82
An excellent antibody against PEX14p, a peroxisomal membrane protein of the
docking complex (Figure 13), was used to analyze the distribution of peroxisomes in the
different fractions obtained after differential centrifugation and density gradient
centrifugation in Western blots.
PEX14p-labelling was very specific, except in the fraction P1 with the cell
debris, revealing the high quality of the antibody used. The main protein band was
found at the expected molecular weight of 60kDa, as expected for PEX14p (Grant et al.,
2013). Peroxisomes were enriched in P3, the typical fraction where most of the
organelles were found. This fraction is frequently called enriched peroxisomal fraction
and corresponds to the “light mitochondral” fraction, since it also contains the small
mitochondria. In comparison, the heavy mitochondrial faction (P2), exhibiting also
large peroxisomes, is less intensively labelled as well as the microsomal fraction (P4),
in which very small peroxisomes were described by electron microscopy (Baumgart,
1997). The strong labelling of the supernatant S4 suggested that during the isolation
procedure of peroxisomes from TM4 cells, some peroxisomes broke. The stronger
peroxisomal enrichment in the gradient is found in fraction B7 and B8. Broken
organelles were also observed in the soluble fraction at the top of the gradient, indicated
by some degradation bands in addition to the PEX14p signal.
Attempts to localize AKAP220 in different subcellular fractions with the antiAKAP220 antibody (Santa Cruz Biotechnology, Inc.) did not give any satisfactory
results. The antibody detected a plethora of different cross-reactive protein bands, but
the expected 220kDa band was labelled very weakly. The strongest signal intensity was
observed for most of the bands in S4, the final supernatant with cytoplasmic proteins.
Based on the results from the Western blot analysis and the peculiar single
localization of the short rat AKAP form into a separate group in the phylogenetic tree
(Figure 17), it appears that the results described by Scott and colleagues (Lester et al.,
1996) with the antibody made against the C-terminal part of the rat AKAP11 protein
might be incorrect. Indeed, careful analysis of the data presented in the paper revealed
several discrepancies. First, they used the TM3 and TM4, which are mouse Leydig cell
line and mouse Sertoli cell line, respectively (Harrison et al., 2001), as rat testis cell
lines to test for rat AKAP220 subcellular distribution study. Second, the mRNA of the
rat AKAP11short from their study was 9748bp in length [U48288.1] containing a 5’untranslated region (UTR) of 5406bp and an open reading frame (ORF) of 3387bp that
Page | 83
encodes a 1129 amino acid polypeptide. However, analysis of this mRNA [U48288.1]
showed that it contains a 5’-UTR of 5411bp and the position of ORF is 5411-8800
(Figure 29). Third, for the expression of a partial AKAP220 of 368 amino acids in E.
coli, they amplified a 1104-bp fragment by PCR, which was encompassing the coding
region of the original AKAP220 cDNA flanked by artificially NdeI and BamHI sites at
the 5’ and 3’ ends, respectively. However, alignment of this primer pair with the
AKAP220 mRNA [U48288.1] showed the forward primer aligned at the position of
8483 and the reverse primer at the position of 8950, which produced an only 467bp
amplicon and 150 bases out of the ORF. Therefore, this incorrect primer pair is assumed
to have negative effect on their analysis.
4.4 Analysis of AKAKP11 (=AKAP220) cDNA with genomic
sequences
Since the commercial anti-AKAP11 rat antibody was not specific against its
target protein, there was a plan to generate a self-made antibody. For this purpose, we
searched for suitable fragments of this protein as the antigen epitope residues by
screening the gene and protein databases for AKAP11. However, the alignment of
various AKAP11 cDNAs showed that sequences from human [NM_016248.3], mouse
[NM_001164503.1] and rat long isoform [NM_012773.2] are highly conserved. It also
revealed that the rat short isoform [U48288.1] does not obey the conservation rule of
AKAP11 evolution. Therefore, it was determined to further analyze the correlation of
the Akap11’s cDNA/mRNA and genome (Figure 27).
Page | 84
ORF: 176-5881
ORF: 202-5886
ORF: 7-5685
ORF: 5411-8800
Figure 27 BLAST Results of Akap11 mRNAs from Genome Database of human [NM_016248.3], mouse
[001164503.1], rat (long [NM_012773.2] and short [U48288.1]), respectively. The exons in red are the
identical sequence in both distinct rat Akap11 mRNAs. The lower short form was published by Scott
group and the protein was suggested to be localized in peroxisomes.
Page | 85
The first AKAP220 antibody was made by Prof. Scott’s group and published in
1996 in the Journal of Biological Chemistry, where the rat AKAP220 protein short
isoform was identified. But from Figure 27, the existence of AKAP11_rat-short mRNA
[U48288.1] and protein isoform [Q62924.1] are questionable.
By BLAST searching the Akap11-human mRNA [NM_016248.3] against the
Human Genome Database①, the exons of the Akap11-human mRNA were localized on
chromosome 13 [NW_001838073.2]. By BLAST searching the Akap11-mouse mRNA
[NM_001164503.1] against the Mouse Genome Database②, all exons of the Akap11mouse mRNA were localized on chromosome 14 [NT_039606.8]. Similarly, by BLAST
searching Akap11-rat-long isoform mRNA [NM_012773.2] against the Rat Genome
Database
③
, all the exons of Akap11-rat-long form mRNA were localized on
chromosome 15 [NW_003811910.1]. Furthermore, [NM_012773.2] and [NP_036905.2]
are mentioned as corresponding mRNA and protein on the Akap11 tag [Rattus
norvegicus (Norway rat)] (Gene ID: 498549, updated on 1-Feb-2015) ④ in the NCBI
Gene database.
However, when a BLAST search was performed for the Akap11-rat-short form
mRNA [U48288.1] originally published by the Scott group, against the Rat Genome
Database as above, the Akap11-rat-short form mRNA was divided into three parts. The
1st part of the mRNA sequence [U48288.1] was identified on chromosome 2
[NW_003806694.1], the 2nd part on chromosome 12 [NW_003811086.1], and the 3rd
part on chromosome 15 [NW_003811910.1]. Also, our analysis showed that the
putative mRNA [U48288.1] was not only including exons from other genes, but also
introns (survival motor neuron protein [NM_022509.1] and EIA-binding protein p400
[NM_001107149.1]). In addition, there were two smaller fragments (2724-2745 and
4822-4842, in orange) identified that couldn’t be found on any chromosome (Figure
27). According to the definition in the book “Gene Cloning and Manipulation” (Howe,
2007), these two fragments contain EcoRI linkers (Figure 28)
①
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=OG
P__9606__9558&LINK_LOC=blasthome
②
③
④
http://www.ncbi.nlm.nih.gov/gene/498549#genomic-regions-transcripts-products
Page | 86
Figure 28 The sequence in the orange frame shows two potential EcoRI linkers in mRNA [U48288.1].
Sequence alignment was done between the position of (2723-2746) and (4821-4843) in [U48288.1].
Taken together with the other identified mistakes, the report of Scott and
colleagues (Lester et al., 1996) described an artificial recombinant clone of Akap11 that
does not exist in nature, in which clone three different genes were fused together via
EcoRI-linkers during the cDNA library construction.
4.5 Northern blotting
To analyze alternatively spliced variants of AKAP11 and to prove that the short
form AKAP11 [Q62924.1] is indeed not existing in rat testis or pituitary gland contrary
to previous report from Scott and colleagues (Lester et al., 1996), it was planned to
detect the expression of all possible isoforms of Akap11 [NM_012773.2] and
[U48288.1] in different rat tissues by Northern blot hybridization.
4.5.1 Total RNA preparation for Northern blotting
Total RNA was isolated from the following rat tissues: testis, brain, lung,
kidney, heart, liver, skeletal muscle, spleen, pituitary.
Table 17 Total RNA was isolated using combined methods, and the RNA quality was checked by RNAChip analysis
Number Sample (date)
Tissue weight
RNA Concentration
Volume A260/280 A260/230
1
Brain-V (18.04)
60mg
307.4 ng/µl
50 µl
2.08
2.18
2
Liver-V (18.04)
60mg
2148.2 ng/µl
50 µl
2.03
2.04
3
Muscle-V (18.04)
60mg
529.5 ng/µl
50 µl
2.08
2.24
4
Liver-V (17.04)
80mg
562.9 ng/µl
80 µl
2.04
1.57
5
Kidney-V (17.04)
80mg
340.9 ng/µl
80 µl
2.10
2.03
6
Spleen-V (18.04)
80mg
1276.4 ng/µl
80 µl
1.95
2.20
7
Pituitary-II (24.04)
20mg
445.9 ng/µl
50 µl
2.08
1.72
Page | 87
8
Testis-V (24.04)
80mg
884.9 ng/µl
50 µl
2.09
2.18
9
Heart-V (18.04)
80mg
111.5 ng/µl
80 µl
2.13
1.66
11
Brain-V (17.04)
80mg
164.3 ng/µl
80 µl
2.08
2.04
12
Pituitary-III (24.04)
20mg
190.6 ng/µl
50 µl
2.10
1.86
14
Muscle-V (26.04)
60mg
153.7 ng/µl
30 µl
1.77
2.25
15
Liver-II (05.03)
20mg
545.9 ng/µl
40 µl
2.07
2.06
16
Testis-V (17.04)
80mg
378.1 ng/µl
80 µl
2.11
2.00
17
Spleen-II (05.03)
20mg
211.1 ng/µl
40 µl
2.08
1.82
19
Testis-II (06.03)
20mg
243.5 ng/µl
40 µl
2.10
1.70
20
Brain-II (05.03)
20mg
118.9 ng/µl
40 µl
2.08
1.39
22
Liver-V (18.04)
80mg
816.4 ng/µl
80 µl
2.08
1.77
23
Testis-II (05.03)
20mg
476.6 ng/µl
40 µl
2.07
1.55
25
Kidney-V (09.05)
80mg
128.0 ng/µl
30 µl
1.77
2.52
27
Brain-V (09.05)
20mg
84.3 ng/µl
40 µl
2.08
1.37
29
Spleen-V (09.05)
20mg
211.1 ng/µl
40 µl
2.08
1.82
30
Lung-V (17.04)
80mg
281.9 ng/µl
40 µl
2.08
2.17
4.5.2 Specific RNA probe generation and validation
The mRNA of Akap11short [U48288.1] has a length of 9748nt comprising both
the 3’- and 5’-UTRs. The ORF is located between nucleotide 5411 and 8800 of the
mRNA sequence. The 3’-UTR is so long that is rare in nature (>5.4kb). The mRNA of
Akap11long [NM_012773.2] has a length of 6950nt including the 3’- and 5’-UTR
regions. The ORF is located between nucleotide 7 and 5685 of the mRNA sequence.
The two Akap11 mRNAs share a common region with 99% identity with 8 gaps
(position 4843-8864 in Akap11short [U48288.1], and position 1608-5633 in Akap11long
[NM_012773.2]).
Page | 88
After a search at the GenomeCube co., one Akap11 EST clone was
subsequenced to be 100% identical with the fragment from 1161 to 4222 in Akap11long
[NM_012773.2], also including a partial common region (1608 to 4222). This clone was
used to generate the specific probes for Northern blot hybridization to identify eventual
Akap11 alternative splicing (Figure 29).
Figure 29 Schematic illustration of the alignment and position of Akap11 rat mRNA [NM_012773.2],
[U48288.1], Akap11 EST clone3 vector and designed primers. Four fragments from the AKAP11
clone3 plasmid that could be used for hybridization probes generation are LSP (230bp), BSP1 (308bp),
BSP2 (893bp) and BSP3 (608bp). LSP: Long Akap11 specific probe; BSP: Both Akap11 specific
probe; NC: negative control.
Page | 89
Figure 30 Constructed plasmids maps, including Akap11 rat mRNA specific hybridization probes LSP (230bp), BSP1 (308bp), BSP2 (893bp) and BSP3 (608bp) and
housekeeping gene probes, such as GAPDH (417bp), β-actin (120bp), 28S rRNA (254bp), and StAR (292bp).
Page | 90
Figure 31 Plasmids linearization for in vitro transcription. C: supercoiled plasmid; -L: antisense
linearized plasmid to generate antisense RNA probes; +L: sense linearized plasmid to generate sense
RNA as positive template control. Plasmid size: LSP (3247bp), BSP1 (3325bp), BSP2 (3910bp), BSP3
(3625bp), GAPDH (3435bp), β-actin (3138bp), StAR (3309bp), 28S rRNA (3271bp), AKAP11 clone3
(7170bp). The 1kb DNA ladder (Promega) was used as the DNA marker
All vectors were linearized with appropriate restriction enzymes and
digoxigenin-labelled RNA probes generated by in vitro transcription as described in
methods (see subsection 3.7.1). The quality of the hybridization probes was verified
using dot blot assay to estimate RNA-probe concentration and DIG-labeling efficiency,
prior to in situ hybridization.
Figure 32 Sensitivity of DIG-labeled antisense RNA probes were verified by dot blot assay.
Page | 91
The intensity of the DIG-labeled probe spots depends on the yield of probe
synthesized during the labeling reaction during in vitro transcription. It depends on the
amount, size, and purity of the DNA template (linearized plasmids shown in Figure 31).
By detecting of the DIG-labeled RNA with a dot blot, also the detection system,
antibody and developing agent were tested. Gradient dilutions of DIG-labeled RNA
were spotted on a positively charged nylon membrane (Figure 32). After heat- and UV
crosslinking of the RNA on the membrane, immunological detection was done by using
a mouse anti-DIG-antibody in combination with a goat anti-mouse IgG secondary
antibody labelled with alkaline phosphatase and subsequent enzyme staining. Our
probes could detect 1-2.5pg RNA, which is sufficiently sensitive for a Northern blot①.
Concerning the specificity and sensitivity of this technique, “rare mRNA can be
detected in 0.1µg of total RNA” by the DIG-labelled non-radioactive Northern blotting
method (DIG Northern Starter Kit Instruction Manual, Roche)②.
8a
8c
8b
8d
Figure 33 Test of 9 different types of washing buffer with 2 distinct dilutions of anti-DIG concentration
①
https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/DIG_AN1_03.1
2.pdf
②
http://post.queensu.ca/~chinsang/lab-protocols/handbooks-and-manuals/dignorthern.pdf
Page | 92
The recipes of 9 types of washing buffers are shown in Table 3, and the
combined 2 kinds of anti-DIG concentration are High anti-DIG (60ng anti-DIG/ml
antibody buffer) and Low anti-DIG (4ng anti-DIG/ml antibody buffer), respectively.
When the washing buffer 8 was used, the membrane 8a appeared as a blank membrane
without the spotted RNA incubated in High anti-DIG as the negative control of
membrane 8c. The membrane 8b was a blank membrane without the spotted RNA
incubated in Low anti-DIG as the negative control of membrane 8d. The membrane 8c
was a membrane containing 1µl of spotted DIG-labelled RNA (4ng) incubated in High
anti-DIG. The membrane 8d was a membrane with 1µl of spotted DIG-labelled RNA
(4ng) incubated in Low anti-DIG. All the membranes were run concurrently using the
different 9 washing conditions and the conditions used for the membrane 8c “Washing
Buffer 8 + High anti-DIG” (with √ mark) yielded the optimal result.
After checking the labelling efficiency in the spot assay, a rat tissue RNA dot
blot was used to test the hybridization efficiency of the DIG-labeled RNA. The
concentration of DIG-labeled RNA probes required was between 20ng/ml (for abundant
mRNA detection, e.g. housekeeping gene) and 100ng/ml (for rare mRNA detection).
Four gradient amounts of total RNA from three different tissues were spotted on
Nylon membranes to analyze the hybridization efficiency, using the probes described in
Figure 32. Hybridization was carried out as described in methods (see subsection 3.7.4).
100ng DIG-labelled Akap11 antisense RNA was used as positive signal control for all
the blots. 1µg non-labelled Akap11 sense RNA was used as positive template control for
LSP and BSP1 blots.
Page | 93
Figure 34 Rat tissue total RNA dot blot hybridization with 4 probes. Left side: methylene blue staining
of the membranes; Right side: corresponding hybridizations. The total RNAs used were from rat kidney,
spleen, testis. The specific RNA hybridization probes used were antisense DIG-labelled RNA of 28S,
Gapdh, Akap11 [NM_012773.2], Akap11 [NM_012773.2] and [U48288.1]. 100ng DIG-labeled Akap11
was used as positive signal control and 1µg non-labeled Akap11 sense RNA was used as the positive
template control for LSP and BSP1.
Dots were visible on the membrane labelled with the antisense RNA probe for
the detection of the 28S rRNA, indicating that the probe bound correctly to the RNAs
from different tissues (Figure 34). Also, the positive signals from the membranes
probed with LSP and BSP1 indicated that the antisense probes bound to the nonlabelled Akap11 sense template correctly. Thus, the absence of signal from the probed
tissue RNAs suggested that the amount of the Akap11 mRNA in the total RNAs was
below the detection limit.
Page | 94
4.5.3 Rat tissue total RNA Northern blot test
Figure 35 Total RNA separation by electrophoresis in a 1.8% formaldehyde-agarose gel using 1×
MOPS running buffer at 20V for 17.5 hours. Methylene blue staining was used to check the transferred
RNA on the membrane, followed by hybridization with the 28S rRNA probe. M: RNA marker
(Promega) 6µl/lane; Lane1: 2# liver 5µg; Lane2: 6# spleen 5µg; Lane3: 3# muscle 3µg; Lane4: 4# liver
3µg; Lane5: 8# testis 3µg; Lane6: 22# liver 3µg (# from Table 17, the RNA quality of these 6 samples
see Chapter 12. Supplemental data S2)
Figure 35 showed that, the whole procedure for non-radioactive Northern
blotting with DIG-labelled probes worked as expected. Different RNA probes were
used to label Northern blots of total RNA isolated from different tissues. However, only
the 28S rRNA control probe hybridization was working well while no positive signals
were obtained from the other probes. There are 2 possible explanations for the
observation. First, the target sense RNA template (e.g. Akap11) is below the detection
limit. In contrast, the 28S rRNA is one of the highest expressed RNAs, therefore its
amount is within the detection limit of the assay. Around 80% of total RNA is rRNA,
15% is tRNA, and some of the rest includes non-coding RNA, thus, protein-coding
mRNA is less than 5% (Lodish et al., 2000). Second, the use of the T7 promoter for the
28S rRNA probe generation supported the efficient in vitro transcription compared to
SP6 promoter used for the other probes (LSP, BSP1, BSP2, BSP3, GAPDH, β-actin,
StAR) (Logel et al., 1992). Suggestion for next trial would be to load more total RNA
(>10µg/well) and to use higher amounts of labelled probes in the hybridization reaction.
Page | 95
4.5.4 Rat total RNA RT-PCR
Because the detection of different Akap11 mRNA form with LSP and BSP1
using the Northern blot did not work as expected, and from the the report that that RTPCR detection is more sensitive than Northern blotting hybridization (Fehr et al., 2000;
Streit et al., 2008), the RT-PCR was used to identify the amount of Akap11 mRNA in
total RNA from rat tissues (Table 17).
Figure 36 Distribution profile of Akap11 mRNA in different rat organs. Lane1: 9# Heart; Lane2: 11#
Brain; Lane3: 12# Pituitary; Lane4: 14# Muscle; Lane5: 16# Testis; Lane6: 20# Brain; Lane7: 22#
Liver; Lane8: 25# Kidney; Lane9: 29# Spleen; Lane10: 30# Lung (# from Table 17); Lane11: 1ng
AKAP11 plasmid clone3 used as positive template control for LSP, BSP1, BSP2, BSP3. As described in
Figure 29, LSP probe is in position of 1240-1449, 230bp; BSP1 is 3113-3399, 308bp; BSP2 is 25283399, 893bp, BSP3 is 2528-3115, 608bp.
Akap11 could be detected with all primer pairs in the different tissues (Figure
36). The expression of the RNA was highest in the pituitary RNA, which is logical and
interesting, since the putative false AKAP11 clone from Scott and colleagues was also
isolated from a cDNA library constructed from pituitary mRNA. Additionally, brain,
testis and spleen exhibited relative high amount of Akap11 expression, whereas this
mRNA was only expressed in low amount in heart muscle and in intermediate quantity
in skeletal muscle, liver, kidney and lung.
The primer pair “Akap11-LSP” for the 5’ part of the mRNA did not amplify the
Akap11 product in the same amounts as “BSP1-3”, since an oligo-dT based RT-PCR kit
Page | 96
was used for the cDNA amplification. For a long mRNA as Akap11 mRNA, it is better
to use a random primer based reverse-transcription kit when it is necessary to amplify
the 5’ part of the mRNA. Regardless of the lower sensitivity of amplification, the
pattern of the tissue distribution for the Akap11-LSP primer pair remained similar,
depicting pituitatry, testis, brain and spleen as the tissues with higher expression of
Akap11.
4.5.5 Concentrated total RNA and PCR-generated probes
Since Akap11 mRNA was detectable in RT-PCR, the possibility that Akap11
primers/probes did not work was excluded. The next solution is to precipitate total
RNAs to get concentrated template, or elute it by polyT columns to get pure mRNAs for
Northern blotting.
Table 18 Total RNA precipitation
Sample
RNA concentration
A260/280
A260/230
Brain
416.8 ng/µl
2.01
2.21
Heart
369.7 ng/µl
1.91
2.38
Liver
378.4 ng/µl
2.03
2.00
Lung
360.6 ng/µl
1.90
2.38
Kidney
1024.1 ng/µl
2.08
2.21
Muscle
2648.2 ng/µl
2.08
2.20
Pituitatry
3122.2 ng/µl
2.09
2.17
Spleen
3163.0 ng/µl
1.98
2.26
Testis
3340.4 ng/µl
2.11
2.21
Figure 37 Alternative method of template
generation for in vitro RNA transcription.
PCR product with T7 promotor could be
used as template instead of linearized
plasmid to further produce RNA probes. The
size of PCR products was 23bp longer than
RNA probes, for instance GAPDH = 440bp;
β-actin = 143bp; LSP = 253bp; BSP1 =
331bp.
Page | 97
This is a PCR-based technique for the preparation of RNA-probes. T7 RNA
polymerase promoters were introduced at the 5’-end of gene-specific oligonucleotide
primers enabling direct in vitro transcription of purified PCR-fragments. The amount of
the DIG-labelled transcripts generated from PCR-templates was equivalent to those
derived from a linearized plasmid. Selection of interesting clones can be dramatically
accelerated by avoiding cloning and plasmid preparation steps using the PCR-approach,
avoiding the uncertain promoter problem from TA-cloning vectors. However, the
minimal length of the PCR-fragment should be at least 300bp as shorter probes are less
sensitive (David and Wedlich, 2001). Due to time limitation, this Northern blotting
assay was not continued and the cloning of AKAP1, -4 and -10 was started.
4.6 Basal analysis and over expression of AKAP1, AKAP4, AKAP10
in TM4 cells
The last 3 amino acids of AKAP1, AKAP4 and AKAP10 suggested they may
contain PTS1 signals (Figure 17). Therefore, the following were carried out: 1) the
tissue distribution and differences in expression of the mRNAs for these AKAPs; 2)
cloning of the AKAPs in mammalian expression vectors; 3) to overexpress different
myc-tag versions of the AKAPs in TM4 cells; and 4) analysis of the subcellular
localization of the AKAPs and subsequent effect on peroxisome and mitochondria. Due
to the unavailability of reliable antibodies for the detection of these AKAPs, the N- and
C-terminals myc-tag versions of these AKAPs were used for the overexpression
experiments.
4.6.1 Basic analysis of AKAP1, AKAP4, AKAP10
The putative C-terminal PTS1 “SKL-variant”-tripeptide of mouse AKAP1 is
“ASL”, whereas “ANL” and “TKL” were found in mouse AKAP4 and AKAP10,
respectively. However, it was reported that a PTS1 is not solely consisting of the Cterminal tripeptide (Brocard and Hartig, 2006). From this research group, an on-line
PTS1 predictor was made available for public use①. Therefore, an analysis could be
done by computer programming (see Supplemental data S1) to show the possible
“trend” for a putative PTS1 signal throughout the 3 AKAP family members.
①
http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp
Page | 98
Figure 38 PTS1 prediction of all AKAP1 family members present in different species (For a larger figure,
see Supplemental data S5, color page 156). Light blue bars --- the length of AKAP1 proteins; green block
--- position of PKA-binding domain; red block --- position of predicted PTS1 signal.
Page | 99
Figure 39 PTS1 prediction of all AKAP4 family members present in different species (For a larger
figure, see Supplemental data S5, color page 157). Light blue bars --- the length of AKAP4 proteins;
green block --- position of PKA-binding domain; red block --- position of predicted PTS1 signal.
Page | 100
Figure 40 PTS1 prediction of all AKAP10 family members present in different species (For a larger
figure, see Supplemental data S5, color page 158). Light blue bars --- the length of AKAP10 proteins;
green block --- position of PKA-binding domain; red block --- position of predicted PTS1 signal.
Page | 101
For the prediction, the both verified- and predicted AKAP protein sequences
were included. Because minimal 12 residues of the C-terminus was required as the
query sequence by the online tool, the last 20 amino acids of AKAP protein members
were used for the PTS1 prediction. Indeed, 52% members of AKAP1 and 48% members
of AKAP10 were predicted to be peroxisomal. The prediction for the last 20 amino
acids of mouse AKAP1 [O08715.4] with a putative PTS1 end “-ASL” as well as the
mouse AKAP10 [O88845.3] with “-TKL” were also to be targeted to peroxisomes, but
not their full sequences. Although none of AKAP4 was predicted to be a peroxisomal
protein, the mouse AKAP4 [Q60662.1] with a known PTS1 end “-ANL” was predicted
to be in “twilight zone”.
Based on the prediction result, it appears as the 3 mouse AKAPs (AKAP1,
AKAP4 and AKAP10) may have a chance to target to peroxisomes. Moreover, the data
from EST sources in NCBI showed that mouse AKAP1, AKAP4 and AKAP10 were the
highly expressed endogenous proteins in testis (Figure 41).
①
Figure 41 Akap1, Akap4 and Akap10 distribution profile (approximate expression pattern inferred from
EST sources in NCBI) ①
From Figure 41 based on mouse, Akap1 transcripts are highly expressed in ovary
and testis, which reflects its potential role in reproduction. Akap4 mRNA is expressed
①
AKAP1: http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.2969,
AKAP4: http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.1498,
AKAP10: http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.274404
Page | 102
only in testis, which suggested its specific function in spermatogenesis. Akap10 mRNA
is mainly expressed in connective tissue, ovary, testis and thyroid.
AKAPs mRNA expression profile in mouse testis
(EST profiler from NCBI Unigene)
600
Transcripts per million (TPM)
500
400
300
200
100
0
Figure 42 Akap1, Akap4 and Akap10 mRNA are high expressed in testis.
Among all Akaps mRNA profile in mouse testis, the highest expressed AKAPcandidates were AKAP1, -4 and -10, suggesting their possible roles in male fertility
(Figure 42). In order to experimentally confirm the results from the EST database
searching, pairs of primers were designed to check the expression level of endogenous
Akap1, Akap4 and Akap10 in mouse testis (Figure 43).
Page | 103
Figure 43 Test of designed primers for Akap1, Akap4, Akap10, and their expression level in different
mouse organs. Br: Brain; Li: Liver; Lu: Lung; Ki: Kidney; Te1: Testis from mouse1; Te2: Testis from
mouse2; Te3: Testis from mouse3; S+g: Sertoli cell + germ cell mixture; LC: Leydig cell line MLTC-1;
SC: Sertoli cell line TM4; pla: the constructed plasmid with required gene fragment, used as positive
template control.
The results showed that Akap1, Akap4, Akap10 mRNA were highly transcribed
in mouse testis, and AKAP1 was also highly expressed in brain in agreement with
Page | 104
previous reports (Dagda et al., 2011; Dyson et al., 2008; Hu et al., 2009; Webb et al.,
2008). It was also observed that Akap1 as well as Star mRNA were highly abundant in
mouse seminiferous tubules (mixture of Sertoli cell + germ cells) and the TM4 Sertoli
cell line. This was unexpected since AKAP1 has only been found in Leydig cells in the
male where AKAP1 regulated StAR protein activity in mitochondria (Dyson et al.,
2008). This observation raised several questions such as: What is AKAP1 function in
Sertoli cells? Is it also localized at the mitochondria surface with Star mRNA and
protein? What happens if the protein is mistargeted?
Akap4 and Akap10 mRNAs were apparently only expressed in germ cells in
testis, but not in the TM4 Sertoli cell line or in MLTC-1 Leydig tumor cells. Whether
these AKAPs are expressed at low levels in Sertoli cells or Leydig cells has to be
verified in the future by using isolated primary cells from the testis and higher amounts
of cDNA as input template.
Figure 44 Test expression of
Akap1, Akap4, Akap10 in TM4
Sertoli cell line under the gradient
FSH dose treatment (see Figure
18). Lane1: 5ng corresponding
plasmids were used as positive
template
control
for
PCR
detection. All the PCRs were run
35 cycles.
As described in the introduction (see subsection 1.2.3), the phosphorylation
signal pathway in Sertoli cell is strongly influenced by FSH induction. But the three
Akaps which control the PKA-dependent phosphorylation cascade in TM4 cell line
showed almost no effect by FSH stimulation. Akap4 and Akap10 were hardly detectable
in TM4 cell. Although Akap1 was expressed quite strongly in TM4 cells, its function
and regulation still remains a mystery. Whether the PKA substrate Star and StAR
Page | 105
protein in Sertoli cells are regulated in the same way as in Leydig cells is also a
question.
4.6.2 Cloning and immunolocalization of myc-tagged AKAPs
The myc tag is a 10-amino acid polypeptide tag (NH2-EQKLISEEDL-COOH)
derived from the c-myc protein and it is frequently used for the localization of
recombinant proteins. The myc-tag system is widely used to facilitate the detection or
purification of fusion proteins. Mouse anti-myc antibodies are also well developed for
immunochemistry. Two different types of vectors were used to monitor recombinant
proteins expression with the myc-tag attached to the target protein at either the N- or Cterminus (pCMV-3B, pCMV-5B) (Terpe, 2003). Because the PTS1 signal is at Cterminus of the protein, two kinds of expression vectors were used for immunodetection
to confirm whether a possible PTS1 signal is really functional. In contrast,
mitochondrial targeting signals are frequently localized at the N-terminus of a protein
and cleaved during the import into the mitochondria① (Emanuelsson et al., 2001).
①
http://www.fastbleep.com/biology-notes/31/172/978
Page | 106
BamHI
BamHI
T3 promoter
T7 promoter
myc-GFP
Myc
GFP-3B
pCMV-GFP-3B
4963bp
GFP-3B-pGEM-Teasy
3716bp
GFP
XhoI
XhoI
T7 promoter
SP6 promoter
To expression vector
To cloning vector
BamHI
BamHI
T7 promoter
T3 promoter
GFP-5B
GFP-myc
GFP-5B-pGEM-Teasy
3716bp
GFP
pCMV-GFP-5B
4958bp
XhoI
SP6 promoter
XhoI
T7 promoter
BamHI
BamHI
T7 promoter
T7 promoter
BamHI
T3 promoter
Myc-AKAP1
AKAP1-3B
AKAP1-3B-pGEM-Teasy
AKAP1-3B-pGEM-Teasy-Mut
AKAP1-3B
Myc
AKAP1-pCMV-3B
5604bp
5604bp
Myc
6853bp
AKAP1
SP6 promoter
T3 promoter
XhoI
XhoI
STOP_Mutation
T7 promoter
EcoRI
NotI
SP6 promoter
T7 promoter
STOP 2715 Glu
To cloning vector
PTS1?
XhoI
point mutation
AKAP1-pYX-Asc
IMAGE clone
AKAP1
BamHI
BamHI
BamHI
T7 promoter
T7 promoter
T3 promoter
AKAP1-5B
STOP_Mutation
AKAP1-5B-pGEM-Teasy
AKAP1-5B-pGEM-Teasy-Mut
AKAP1-5B
AKAP1-pCMV-5B
AKAP1
5604bp
5604bp
6849bp
AKAP1-Myc
Myc
SP6 promoter
SP6 promoter
XhoI
PTS1?
XhoI
STOP_Mutation
STOP 2715 Glu
T7 promoter
XhoI
Page | 107
BamHI
T3 promoter
BamHI
T7 promoter
Myc
AKAP4-pCMV-3B
AKAP4-3B-pGEM-Teasy
6829bp
5580bp
AKAP4
Myc-AKAP4
AKAP4-3B
TOPO
SP6 promoter
SP6 promoter
PTS1?
T7 promoter
XhoI
XhoI
To cloning vector
AKAP4-pCR-Blunt II-TOPO
IMAGE clone
AKAP4
BamHI
BamHI
T7 promoter
T3 promoter
T7 promoter
TOPO
AKAP4-pCMV-5B
AKAP4-5B-pGEM-Teasy
AKAP4
6825bp
5580bp
Myc
AKAP4-Myc
AKAP4-5B
P
PTS1?
SP6 promoter
T7 promoter
XhoI
XhoI
EcoRI
T3 promoter
SP6 promoter
Myc-AKAP10
EcoRI
Myc
AKAP10-pCMV-3B
AKAP10-3B-pGEM-Teasy
AKAP10
5416bp
4146bp
AKAP10-3B
XhoI
T7 promoter
SP6 promoter
T7 promoter
NotI
PTS1?
XhoI
To cloning vector
AKAP10
AKAP10-pSPORT 1
IMAGE clone
EcoRI
SP6 promoter
SalI
EcoRI
T7 promoter
T3 promoter
AKAP10-Myc
AKAP10-5B-pGEM-Teasy
AKAP10
AKAP10-pCMV-5B
4146bp
AKAP10-5B
Myc
5412bp
XhoI
T7 promoter
T7 promoter
XhoI
Figure 45 The instruction of myc-tag recombined plasmids generation (inserts are: GFP, AKAP1, AKAP4 and AKAP10)
Page | 108
PTS1?
P
The procedure for subcloning of myc-tagged GFP- and AKAP-fusion proteins
had been described in detail in the methods (section 3.8). To check the localization of
fusion protein in vivo, the critical point is the specific binding of the antibody against
the tag in fusion protein. Two positive control plasmids, pCMV-GFP-3B and pCMVGFP-5B, were constructed to test the functionality of the vectors to express the desired
protein in TM4 cells. The myc-tag was fused to the N-terminus and C-terminus of the
GFP, respectively.
GFP (green fluorescent protein) is a 238-residues green fluorescent protein with
an emission peak at 509 nm, and it is widely used as a reporter of expression (Tsien,
1998). GFP was previously thought to have non- or low toxicity and exhibit no
interference with any biological processes (Olivares-Fuster et al., 2002; Welsh and Kay,
1997). Therefore, many engineering GFP variants were generated for biological
monitoring (Hoffman, 2008; Murphy et al., 2011). However, recently, some side effects
of GFP were reported from in vitro and in vivo studies, such as cytotoxicity and
apoptosis induction (Koike et al., 2013). Thus, in this thesis the myc-tag, which
exhibited small size (only 10 amino acids) and no side effect, was used instead of GFP
to track the fused AKAP proteins.
GFP is a cytoplasm protein with no targeting signal at any terminus, hence the
attached myc-tag should not interfere with its cytoplasmic localization. This
arrangement will also allow the co-localization of GFP and the myc-tag in the both two
constructs, which was confirmed in Figure 46 (myc-GFP and GFP-myc). These two
GFP fusion constructs were also used to optimize the transfection conditions and the
staining conditions for the anti-myc tag antibody labelling.
Page | 109
Figure 46 Co-localization test
for the myc-tagged GFP-fusion
proteins as positive controls,
N-terminal myc-labelled GFP
(GFP-3B) plasmid and Cterminal myc-labelled GFP
(GFP-5B) plasmid. A and B
were taken with a 63 ×
objective of the conventional
fluorescence
microscopy
(Leica DMR). C and D were
taken with a 63× objective of
the confocal laser scanning
microscopy (CLSM) (Leica
TCS SP2).
N-myc-GFP
(GFP-3B) and GFP-Cmyc (GFP-5B) were both
expressed
in
the
cytoplasm
and
also
nucleus, whilst the antimyc
mainly
antibodies
only
stained
the
cytoplasm. These results
can be explained in two
ways. First, the binding
of the antibodies to the
outer region will make the penetration to the interior of the cells and nucleus more
difficult for next sets of antibodies. The second possibility is that the permeabilization
of the nuclear pores after fixation of the cells during the immunofluorescence staining
caused the poor penetration of antibodies into the nucleus, whereas the GFP-myc
recombinant protein was transported in the living cell during the cell culture into the
nucleus prior to the fixation already. The general entry permeabilization conditions for
primary and secondary antibodies into mitochondria with a double membrane (outer and
inner mitochondrial membrane, see Figure 48D-F. SOD2 in the mitochondrial matrix)
and also into peroxisomes with a single limiting membrane (see Figure 48A-C. catalase
staining in the peroxisomal matrix) gave excellent results.
Page | 110
It was suspected that the AKAP1, -4, -10 contain a putative PTS1 targeting
signal at the C-terminus, and AKAP1 and AKAP10 have been suggested to contain a
mitochondrial targeting motif in the N-terminus, it was necessary to visualize
peroxisomes and mitochondria in the TM4 cell line under ordinary growth conditions
(Figure 47 and Figure 48). Peroxisomes are numerous in TM4 cells (Figure 47A-D) can
be well labelled with PEX14p (Figure 47C-D), a protein marker for peroxisomes that
was strongly labelling in Sertoli cells in the testis (Grant et al., 2013; Nenicu et al.,
2007).
Figure 47 Immunofluorescence
images showing peroxisomal
membrane staining (A-D) in
untreated
TM4
cells.
Micrographs were taken with a
63 ×
objective
of
the
conventional
fluorescence
microscopy (Leica DMR).
PEX3p
was
less
expressed and peroxisomal
staining was only possible
with
high
antibody
concentrations, leading to
more background (Figure
47A-B).
TM4
cell
peroxisomes are a mixture
of round, rod-shaped and
elongated,
tubular
organelles that are distributed around the nucleus and sometimes form clusters or small
network-like structures. In the TM4 cell processes, peroxisomes showed a
heterogeneous pattern, with some processes exhibiting many peroxisomes until the end
of the cellular extension (Figure 47C) and others exhibiting only few stretched
organelles (Figure 47A), suggestive of a transport via microtubules in this region.
Page | 111
Figure 48 Immunofluorescence images showing peroxisomal (A-C) and mitochondrial (D-F) matrix
staining in untreated TM4 cells. Micrographs were taken with a 63× objective of the conventional
fluorescence microscopy (Leica DMR).
Staining for catalase provided a similar pattern in TM4 cells as for PEX14p
(Figure 47C-D). Catalase was sometimes more enriched in the large, round and
peripheral part of tubular peroxisomes (bright end) whereas less staining of the tubules
in between was noted, which could even be better identified in less intensively stained
peroxisomes (Figure 48B-C). Large clusters and network-like structures were also
visible with the catalase staining (Figure 48A and C). Mitochondria were well stained
with the anti-SOD2 antibody in TM4 cells (Figure 48D-F). Large differences were
observed between the different TM4 cells in the same culture, with some cells
exhibiting very long mitochondria in the cell periphery, which seem to extend from a
perinuclear mitochondrial reticulum. Similar to catalase also SOD2 seems to be
enriched in some matrix parts in the very long mitochondria (Figure 48D). In other TM4
cells (Figure 48E), mitochondria are arranged in a complete network up to the leading
lamella of the cell (insert in Figure 48E: perinuclear mitochondrial network). In the
Page | 112
backward pole with an elongated extension, single long mitochondria were stretched
underneath the plasma membrane and seemed to be transported along microtubules.
Some TM4 cells contained a very dense mitochondrial network with rather small
distances and high levels of SOD2 (Figure 48F).
Figure 49 Double-immunofluorescence images of the myc-tag and PEX14p, revealing that both myctagged AKAP1 fusion proteins did not co-localize with peroxisomes. Micrographs were taken with a
63× objective of the conventional fluorescence microscopy (Leica DMR).
Inhibition of the mitochondrial targeting with the N-terminal myc-tag did not
promote the targeting of the recombinant AKAP1 protein to peroxisomes (Figure 49AC). In some cases, larger and nicely extended cells were observed in the cell cultures.
These cells did not exhibit a high overexpression of the recombinant N-myc-AKAP1
protein. N-myc-AKAP1 was observed in a network-associated pattern without an
association with peroxisomes (see Figure 49C), although peroxisomes were always
Page | 113
located at the areas of weakly labeling N-myc-AKAP1. Since some parts of the nuclear
membrane seemed to be positively labelled with the anti-myc antibody (Figure 49C),
the association of N-myc AKAP1 with the cytoplasmic face of the endoplasmic
reticulum could be possible, but this has to be proven in the future with anti-ER-marker
antibodies. In cells overexpressing AKAP1-C-myc, a typical mitochondrial pattern was
observed. With the C-terminal myc-tag, a putative peroxisomal targeting would be
blocked, since the PTS1 must be a “free” tripeptide signal at the extreme C-terminus to
ensure the targeting of proteins into the peroxisomal matrix. In these cells, however,
some peroxisomes exhibited very extended elongated structures with a size and
appearance similar to mitochondria (see Figure 49F).
Figure 50 Double-immunofluorescence showing that N-terminal myc-tagged AKAP1 (AKAP1-3B) did
not co-localize with mitochondria (Figure 50A-C), but C-terminal myc-tagged AKAP1 (AKAP1-5B)
co-localized with mitochondria (Figure 50D-F). Micrographs were taken with a 63× objective of the
conventional fluorescence microscopy (Leica DMR).
Page | 114
Also, in the co-localization staining for mitochondria, the recombinant AKAP1
protein with a myc-tag at the N-terminus (N-myc-AKAP1) exhibited a cytoplasmic
localization with enrichment in some areas of the periphery, whereas the AKAP1
protein with a C-terminal myc-tag (AKAP1-C-myc) was targeted to mitochondria, as
shown by the overlapping of myc and SOD2 stains. In cultures with overexpressed Nmyc-AKAP1, a clear downregulation of SOD2 was noted in transfected cells, whereas
some untransfected cells exhibited a much stronger SOD2 labelling, suggesting a
difference in the reaction to oxidative stress between transfected and non-transfected
cells. AKAP1-C-myc targeted to all mitochondria in a similar level, whereas the SOD2
was clearly localized only in some parts of these mitochondria. The insert in Figure 50F
depicts a TM4 cell with ring-shaped mitochondria, in which the SOD2 was also
heterogeneously localized. The overall heterogeneity of SOD2 localization in the
mitochondria of “lipofected” TM4 cells seemed to be much higher than in regular
control cells without any transfection (compare to Figure 48D-F). At higher
magnifications, the punctate mitochondrial pattern could be identified in some cases as
longer organelles, only labelled strongly at some parts with SOD2, forming “beads on a
string”-like patterns inside the cells. The mitochondrial network in cells with high
overexpression of AKAP1-C-myc was very dense and exhibited small organelles.
CLSM examinations of the immunofluorescence preparations revealed similar
results as obtained by conventional fluorescence microscopy, therefore only the
overlays of different examples of transfected cells are presented.
Page | 115
Figure 51 CLSM images of double-immunofluorescence for myc and SOD2,
showing that N-terminal myc-tagged AKAP1 (AKAP1-3B) did not co-localize with
mitochondria (Figure 51A-B), unlike the C-terminal myc-tagged AKAP1 (AKAP15B) (Figure 51C-D). Micrographs were taken with a 63× objective of the confocal
laser scanning microscopy (Leica TCS SP2).
The overexpressed recombinant N-myc-AKAP1 protein clearly localized to
special parts of the cytoplasm and network-like structures (Figure 51A), partially
covering the nucleus (Figure 51B), suggesting that certain parts of the ER-system could
be labelled. The mitochondrial network in N-myc AKAP1 overexpressing cells seemed
to be slightly less extended to the periphery and more centrally localized in the
perinuclear area. Moreover, the long mitochondria observed in the cytoplasmic region
underneath of the nuclear region of non-transfected neighboring cells (Figure 51A) were
Page | 116
less frequently observed in transfected cells. Mitochondria were localized in N-mycAKAP1 free areas, sometimes closely covered by N-myc-AKAP1 positive structures
(Figure 51A & B). In strong AKAP1-C-myc overexpressing cells, AKAP1 was targeted
to the complete mitochondrial population, whereas SOD2 was heterogeneously
distributed, therefore in addition to the yellow color indicative of the overlapped stains,
red and green colored mitochondria were clearly visible at higher magnifications
(Figure 51C). In very strong AKAP1-C-myc overexpressing cells, mitochondria were
rounded up and single organelles or packaged in a very dense network. Cells with lower
expression of AKAP1-C-myc exhibited already proliferated mitochondria and a denser
network than non-transfected TM4 cells. In addition, they exhibited a strong SOD2
staining of the entire mitochondrial network, therefore they appeared green even though
a weak red staining of the low expressed AKAP1-C-myc in mitochondria was present
(Figure 51D).
Figure 52 Double-immunofluorescence preparations for myc and SOD2, showing that the two types of
myc-tagged AKAP4 were both expressed in the cytoplasm and did not co-localize with mitochondria in
TM4 cell line. Micrographs were taken with a 63 × objective of the conventional fluorescence
microscopy (Leica DMR).
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Both overexpressed recombinant AKAP4 proteins were localized in the
cytoplasm and reduced the abundance of mitochondrial SOD2 in transfected cells. In
addition, the level of SOD2 was high in the mitochondria of neighboring TM4 cells that
were not transfected with the AKAP4-C-myc protein.
Figure 53 CLSM images of double immunofluorescence preparations for myc and SOD2, showing that
the both types of myc-tagged AKAP4 fusion proteins localized within the cytoplasm but not to
mitochondria in TM4 cell line. Micrographs were taken with a 63× objective of the confocal laser
scanning microscopy (Leica TCS SP2).
Cells expressing intermediate levels of AKAP4 in the cytoplasm contained a
more centrally localized mitochondrial network with lower levels of SOD2, especially
when the N-myc AKAP4 was overexpressed. Only two positive cells were found
overexpressing AKAP4-C-myc. The first one exhibited a collapsed perinuclear
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mitochondrial network with only very few mitochondria extrusion (Figure 53C). The
second cell appeared more normal, however, exhibited lower SOD2 levels than the
neighboring non-tansfected cells.
Figure 54 Double-immunofluorescence preparation for myc and PEX14p, showing that N-terminal
myc-tagged AKAP10 (AKAP10-3B) did not co-localize with peroxisomes. Micrographs were taken
with a 63× objective of the conventional fluorescence microscopy (Leica DMR).
In the N-myc AKAP10 overexpression experiments some giant cells were
present. All cells exhibited a mainly cytoplasmic N-myc-AKAP10 staining, with some
positive small and round organelles (Figure 54C). These dots did not colocalized with
peroxisomes that were labelled for PEX14p (Figure 54C). The overall peroxisomal
appearance and distribution pattern in these cells was also not altered. Most of the cells
exhibited round to rod-shaped peroxisomes and less tubules.
Figure 55 Double-immunofluorescence preparation for myc and SOD2, showing that the N-terminal
myc-tagged AKAP10 (AKAP10-3B) did not co-localize with mitochondria. Micrographs were taken
with a 63× objective of the conventional fluorescence microscopy (Leica DMR).
Page | 119
N-myc-AKAP10 was also localized in the cytoplasm, but did not lead to the
reduction of mitochondrial number or the reduction of SOD2. AKAP10-C-myc
overexpression seemed to be toxic since no positive transfected cells within 24 hours.
The transfected cells probably died due to apoptosis, which might have been induced by
altered mitochondrial function. To determine whether N-AKAP10 would have an effect
on mitochondria and apoptosis induction, therefore earlier time-points after the
transfection have to be used in future experiments.
Figure 56 CLSM images of a double immunofluorescence preparation for myc and SOD2, showing
that the N-terminal myc-tagged AKAP10 (AKAP10-3B) did not co-localize with mitochondria.
Micrographs were taken with a 63× objective of the confocal laser scanning microscopy (Leica TCS
SP2).
In comparison to neighboring non-transfected cells, cells with low N-mycAKAP10 expression in the cytoplasm exhibited a less dense mitochondria network and
a lower SOD2 labelling. The contrast of the AKAP10 and SOD2 staining was enhanced
to aid better visualization of the mitochondrial network in the insets (Figure 56A). Cells
with high N-myc-AKAP10 expression most probably went into apoptosis (round cell in
Figure 56B with only few mitochondria).
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5 Discussion
5.1 AKAP220 (-CRL) is probably an artifact
The A-kinase anchoring proteins (AKAPs) constitute a protein family that tether
PKA and other enzymes to various specific subcellular locations, and play a critical role
in the regulation of the cellular physiological metabolism. In 1996, a rat isoform of
AKAP11 which was previously called rat AKAP220 [Q62924.1] due to its molecular
mass, was reported to be localized in peroxisomes through a PTS1 signal (“-CRL”) and
supposed to take part in testicular peroxisomal functions and phosphorylation events by
Prof. Scott’s group, an AKAP research pioneer (Lester et al., 1996). As a peroxisome
research group, this particular “peroxisomal” AKAP was interesting for us. However,
during this thesis we revealed that this putative peroxisomal rat AKAP11 [Q62924.1] is
an artifact, that was mistakenly identified in the literature.
First, a functional structure of a protein should be evolutionary conserved
throughout the entire family of the protein (Nasir et al., 2014). It is unlikely that just one
member possesses some functional domain not present in any other members of related
species. From the information obtained from the literature and the screening of all
AKAPs (Figure 17), we found that the reported sequence of rat AKAP11 [Q62924.1] is
the only one with a distinct C-terminal end containing a putative PTS1 signal in the
entire AKAP11 family, and clusters into a separate protein subfamily in the
evolutionary tree.
Second, the result of the mRNA BLAST showed that the mRNA [U48288.1] of
rat AKAP11 [Q62924.1] is not derived from a single chromosome, but combines three
non-related gene fragments from three distinct chromosomes by two EcoRI linkers
(Figure 27-28). In the 1990’s, EcoRI linker was frequently used for self-made cDNA
library constructions, which could have been the source of the contamination. Although,
“trans-splicing” events exist in nature that mRNA might be not 100% derived following
exons order in genome (Caudevilla et al., 1998; Flouriot et al., 2002; Yang and Walsh,
2005). However, one mRNA from 3 separate chromosomes (2, 12 and 15) is very
unlikely (Figure 27). Moreover, in the right exon from chromosome 15, an artificial
frame shift (position: 8864-9726) in the cDNA clone caused an upper STOP codon,
leading to the “-CRL” PTS1 signal.
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Third, the antibody against AKAP220 used in Scott’s paper was produced by the
cloned artificial recombinant partial AKAP220 plasmid. Using this antibody, a double
immunofluorescence staining showed the peroxisomal localization of AKAP220.
Moreover, the antibody used in the Scott’s paper is polyclonal, meaning that this
antibody was composed of an antibody mixture targeted to different epitopes on rat
AKAP220. This mixture most likely also contained antibody that recognized the
tripeptide (“-CRL”) in the C-terminus of rat AKAP220. Antibodies against the Cterminal PTS1 signal, such as “-SKL”, are frequently used to stain peroxisomal protein
efficiently and will clearly label many proteins in the peroxisomal compartment (Stelzig
et al., 2013). The “-CRL” could also be potentially found at the C-terminus of other
peroxisomal proteins which could be then detected by this antibody. Therefore, during
the bioinformatics work for this thesis, the complete protein database of the rat was
searched for a “CRL-STOP codon”. Indeed, with the advice from the Swiss Institute of
Bioinformatics, a rat peroxisomal protein Acot4 [D3ZIQ1] (421aa, 46kDa) was found
with a “-CRL” tripeptide at the C-terminal end. This could be an explanation for the
detected peroxisomal staining for rat AKAP220 presented in Scott’s paper.
We therefore suggest that the rat AKAP220 [Q62924.1] which was described to
be a peroxisomal protein because of its “-CRL” putative PTS1 signal, is an artifact,
resulting from a non-accurate self-made cDNA library construction. This hypothesis is
practically proved by our findings of the EcoRI linker at two different positions inside
of the rat AKAP11 recombinant cDNA clone (Figure 28). Similar mistake was common
in that period, due to technical limitations and the lack of the shared database
information that we are able to access nowadays. For example, a shorter cloned mouse
AKAP10 gene was generated also due to an artificial cDNA library or false sequencing,
causing a frame shift and a wrong STOP codon (Wang et al., 2001).
5.2 PTS1 prediction is useful for searching novel peroxisomal
proteins
The peroxisomal enzyme composition in distinct organs and cell types is
heterogeneous and varies strongly (Grant et al., 2013). Due to the limitation in pure
peroxisome isolation from different cell types, the peroxisome proteome research still
depends on the computational prediction to identify low abundance peroxisomal
proteins. The C-terminal PTS1 tripeptides (“-SKL” variants) are the most important part
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of the PTS1 signal and widely used as the first strategy to screen for potential new
peroxisomal matrix proteins (Hayashi et al., 1997; Ma and Reumann, 2008).
However, the larger the database tested or the bigger the PTS1 tripeptides
pattern matrix applied, the more candidates would be generated as well as the more
false positive hits obtained. Because this screening algorithm excludes the analysis of
other critical factors on the individual candidate, such as 1) upstream amino acid
residues close to the PTS1 tripeptides, 2) other possible organelle targeting signals
ahead or overlapping the PTS1 signal, and 3) the unpredictable C-terminal exposure
efficiency. In 2008, a study applied a PTS1 tripeptides pattern matrix ([SAPC]-[KR][LMI]) to screen an Arabidopsis protein database and obtained 7 potential plant
peroxisomal kinases from that screening. However, 4 candidates were verified with
functional PTS1 only when they were partially expressed as oligopeptides, but not as
full-length fusions which remained in the cytoplasm. The other 3 of the 7 sequences
were incorrectly predicted through experimental validation (Ma and Reumann, 2008).
Also a mammalian PTS1 motif was proposed ([SACKN]-[KRHQNS]-[LI]) (Amery et
al., 2000). In this thesis, we applied a broad-ended PTS1 consensus ([ACHKNPQST][AHKNQRS]-[FILMV]) and a test database for AKAPs, and obtain 3 AKAP families
with potential PTS1 signals which were highly expressed in the testis (Table 15 and
Figure 17&41-42).
Furthermore, our second screening which included the upstream residues of the
PTS1 terminus suggested a low possibility of peroxisomal proteins throughout these 3
AKAP families (Figure 38-40). Basic residues located close to PTS1 tripeptides play an
enhancing role for the peroxisomal targeting, while acidic amino acids are functionally
inhibitory instead, especially the critical residue at the 4th last position (Amery et al.,
2000; Ma and Reumann, 2008). For example, a GFP construct derived from human 2methylacyl-CoA racemase, which coded GFP-KVKASL, localized very well in
peroxisomes. Amery and colleagues showed that a nonpolar side chain amino acid point
mutant GFP-KVGASL displayed a cytoplasmic fluorescence (Amery et al., 2000).
In this respect, a closer look on the 3 individual mouse AKAPs was taken:
The mAKAP1 [O08715.4] contains the reported PTS1 variant “-ASL” (Amery
et al., 2000), but without positive charged Lysine (Lys, K) at the 4th last position, on the
contrary one negatively charged Aspartic acid (Asp, D) and 3 uncharged amino acids
(Ser-Tyr-Tyr, SYY) upstream were found which might be inhibitory on the peroxisomal
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targeting signal (Figure 38). In addition, mAKAP1 also contains a mitochondrial
targeting signal in the N-terminal 30 residues, which maintains higher priority and is
exposed first during translation.
Eventhough mAKAP4 [Q60662.1] also contains a prototypical PTS1 tripeptide
“-ANL”, the presence of one Aspartic acid (Asp, D) and 3 hydrophobic amino acids
(Trp-Leu-Leu, WLL) upstream might reduce the peroxisome targeting ability (Figure
39).
The C-terminal end “-TKL” in mAKAP10 [O88845.3], considered as a putative
PTS1 signal here (Figure 40), has been suggested to act as typical class I PDZ-binding
motif (“-STKL”) conserved among species (Tingley et al., 2007). PDZ domains are
abundant and diverse and mediate protein-protein interactions. More than 200 types of
structures are present to recognize the C-terminus of binding proteins, such as the Cterminal end of Shaker-type K+ channels (Kvl1.4) and N-methyl-D-aspartate receptor 2B
(NMDA) (Lee and Zheng, 2010; Saras and Heldin, 1996). The PDZ-binding motif can
serve as signal peptide to target proteins to membrane receptor complexes (Hamuro et
al., 2002). Moreover, AKAP10 was shown to localize at a highly specialized region of
the plasma membrane in mouse kidney proximal tubular cells. This PDZ-binding motif
in AKAP10 could be recognized by PDZ domain-containing proteins, which might
influence phosphorylation of PKA substrates located in close proximity to the plasma
membrane. For example, PDZK1, a protein with multiple PDZ domains, connects
plasma membrane proteins and acts as an anchoring site for AKAP10 via its PDZbinding motif (Gisler et al., 2003a; Gisler et al., 2003b). Thus, the putative PTS1 signals
of these 3 AKAPs can hardly be functional under non-pathological conditions.
The on-line PTS1 predictor in the second round revealed that the mouse
AKAP1, 4, 10 are probably not the targeted to peroxisomes (Figure 38-40), which was
also confirmed by overexpression of AKAP1-pCMV-3B, AKAP4-pCMV-3B and
AKAP10-pCMV-3B in this study (Figure 49-56). Thus, this web tool seems to be
relatively reliable to predict the targeting possibility. In addition, a combined computer
programming code (see Supplemental data S1) was developed for large amount
sequence screenings. The programming languages named Python and R were used, both
of which are easy to learn for beginners and are open-source free software for
worldwide users (Prechelt, 2000; Tippmann, 2015). In this study, Python was used for
screening PKA binding domain and PTS1 signals throughout the whole AKAP1, 4, and
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10 family members in the protein database, and R was used for transferring results into
pictures to be easily read. The code (Supplemental data S1) is free offered for any user,
who has interest to have a predicted overview of sequence feature before executing
experiments.
5.3 Exceptions to the rules of PTS1 prediction: alternative targeting
of peroxisomal matrix proteins
Not all the sequences indicated by the PTS1 predictor are peroxisomal protein.
Also, some peroxisomal proteins did not follow the general algorithm of defined PTS1
prediction, such as the proteins that contain “hidden” PTS1 signals, which are only
appended by translational readthrough extension. For instance, lactate dehydrogenase, a
cytoplasmic enzyme, was described in peroxisomes in 1996 (Baumgart et al., 1996a),
however, nobody knew its targeting mechanism to the organelles. Last year, it was
described that lactate dehydrogenase B (LDHB) contains a PTS1 signal at the Cterminal end that cannot be recognized by any available PTS1 prediction programs,
because it is located after the STOP codon. Only when translational read-through
occurs, it will be functional and will target LDHB to peroxisomes (Baumgart et al.,
1996a; Schueren et al., 2014).
Some proteins don’t contain a PTS1 signal, but are also transported into
peroxisomes by binding a partner which contains a PTS1. For example, Cu/Zn
superoxide dismutase 1 (SOD1) is an enzyme without any targeting signal, while its
binding partner, the “copper chaperone of SOD1” (CCS), contains a weak PTS1 (“AHL”). Therefore, SOD1 is co-imported by CCS into peroxisomes via a “piggyback”
import-mechanism. When expressed together, SOD1 will be shuttled into peroxisomes
by interacting with CCS. When a defect in the SOD1-CCS association occurs, for
example by a mutation in the PTS1 end of CCS or in the peroxisomal import receptor
PEX5p, both SOD1 and CCS remain in the cytoplasm (Islinger et al., 2009).
Some proteins can display dual localization under different situations. In rat, Lalanine: glyoxylate aminotransferase 1 (AGT1) is in mitochondria, and its N-terminal
22 residues were considered to be a putative mitochondrial targeting signal. In human,
AGT1 lost these 22 amino acids during evolution and is targeted to peroxisomes.
However, in primary hyperoxaluria type I patients, the AGT1 is transferred again back
to mitochondria, caused by a point mutation in the initiation codon ATG to ATA
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(Takada et al., 1990). A protein with real dual subcellular localization is human 2methylacyl-CoA racemase, which is a protein containing both mitochondrial and
peroxisomal targeting signals (Amery et al., 2000).
5.4 AKAP1 and mitochondrial fragmentation
It was reported in the literature that in many cell types (neurons, cardiomyocyte,
etc.) the overexpression of AKAP1 could promote phosphorylation and inhibition of the
pro-apoptotic BAD protein (Affaitati et al., 2003; Merrill and Strack, 2014). PKA
phosphorylates BAD at Ser155, which blocks BAD binding of Bcl-2 and inhibits
apoptosis. Mitochondrial dysfunction is associated with apoptosis, indicating that
mitochondrial-anchoring of PKA is critical for cell survival (Carlucci et al., 2008).
Moreover, overexpression of AKAP1 and Ser637-phosphorylation of Drp1 was
neuroprotective, promoting mitochondrial elongation by fusion (Cribbs and Strack,
2007; Merrill et al., 2011). In conclusion, high cellular AKAP1 levels were suggested to
promote mitochondrial elongation or aggregation, and to reduce fragmentation or
apoptosis (Kim et al., 2011).
Although it was shown that AKAP1 overexpression is good for the
mitochondrial morphology and cell survival, in our case the extra myc-tag might hinder
it. In this thesis, when AKAP1-pCMV-5B (C-term myc-tagged AKAP1) was overexpressed in TM4 cells, some cells displayed more fragmented mitochondria (Figure
51C-D). It was reported that AKAP1 with a defect in the KH domain induced changes
in the mitochondrial morphology that appeared collapsed and rounded up rather than the
normal elongated rod-like shape (Rogne et al., 2009). When the KH domain is
phosphorylated by PKA, it is active to bind and stabilize RNA or ssDNA, such as Star,
Sod2 and ATP synthase F0-f mRNAs. A KH domain mutant is associated with some
diseases, such as fragile X mental retardation syndrome and paraneoplastic opsoclonusmyoclonus-ataxia (POMA) neurodegenerative syndrome (Rogne et al., 2009). It is
suggested that the C-terminal myc tag of recombinant AKAP1 may spatially hinder the
KH domain phosphorylation or its mRNA association, possibly reducing mitochondrial
protein content and inducing mitochondrial fragmentation.
Moreover, mislocalized AKAP1 reduced oxidative ATP synthesis and
mitochondrial membrane potential (ΔΨm) and increased oxidative stress resulting in
cell death, and the AKAP1 knockdown induced mitochondrial fragmentation and
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apoptosis (Perrino et al., 2010). Thus, AKAP1 was suggested to play an important role
in restoration of mitochondrial respiratory function, reduction of mitochondrial ROS
and reduction of mitochondrial fission. However, in this study when AKAP1-pCMV-3B
(N-term myc tagged AKAP1) was over-expressed in TM4 cells, the cells displayed
normal elongated tubular mitochondria (Figure 51A-B). Additionally, the peroxisomal
morphology remained unaffected regardless of the AKAP that was transfected.
5.5 Mitochondrial fusion and fission mechanisms
The mitochondria fusion and fission machinery is tightly controlled by protein
phosphorylation. Mitochondrial fusion is controlled by three GTPase: Mitofusion1
(Mfn1), Mitofusion2 (Mfn2) and optic atrophy 1 (OPA1). Mfn1 and Mfn2 are anchored
in the mitochondrial outer membrane (MOM), and OPA1 is localized to the
intermembrane space and involved in the control of the cristae structure. The
mitochondrial fission process is regulated by a cytosolic GTPase dynamin-related
protein 1 (Drp1) and a small outer mitochondrial membrane binding protein Fis1. Fis1
is also responsible for peroxisomal division, which is discussed in following section 5.6.
As mentioned in section 5.4, Drp1 is inactivated by PKA phosphorylation at
Ser637, which inhibits its GTPase activity. Inactivated Drp1 promotes elongation of
mitochondria and enhances cellular resistance to pro-apoptotic stimuli. Dephosphorylation of Drp1 phospho-Ser637 by CaN (also called PP2B), an AKAP1
binding partner, induces mitochondrial fragmentation and cell death. When active,
dephosphorylated Drp1 rapidly cycle between cytosol and mitochondria, suppressing
mitochondria fusion into the cell protective reticulum (Merrill et al., 2011). AKAP1 at
the mitochondrial membrane provides docking sites for the C-terminus of Drp1, as well
as the central domain of PKA holoenzyme, which are indispensable for regulating
mitochondrial morphology (Kim et al., 2011). Thus, it could be another possible
explanation for the transfected AKAP1-myc induced mitochondria fragmentation. The
C-terminal myc-tag might block the Drp1 docking site in AKAP1, wherefore, active
Drp1 remain in the cytosol, a process inhibiting mitochondrial fusion and maintaining a
punctate mitochondrial pattern.
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5.6 Connection between peroxisomes and mitochondria
Distinct sets of enzymes are present in peroxisomes and mitochondria for lipid
β-oxidation in animals, the fatty acid degradation still needs the cooperation of both
organelles, because shortened fatty acids from peroxisomes are broken down in
mitochondria (Colasante et al., 2015). For example, the peroxisomal acyl-CoA oxidases
and the mitochondrial acyl-CoA dehydrogeneases originated from a common ancestor
gene (Baumgart et al., 1996b).
The division processes of peroxisomes and mitochondria also influence each
other by shared fission proteins, such as PMD1 (Peroxisomal and Mitochondrial
Division Factor 1), dynamin GTPases DLP1, Fis1 (Fission factor 1) and Mff
(Mitochondrial fission factor). Fis1 promotes both mitochondrial and peroxisomal
division, targets to peroxisomes by a PEX19p shuttle and contacts with PEX11α and
PEX11β (Schrader et al., 2011). Mitochondria and peroxisomes also cooperate in antiviral signaling and defense (Islinger et al., 2012). For example, mitochondrial antiviral
signaling adaptor (MAVS) is also a dual-localized protein. When HIV-RNA attached on
MAVS sites, the MAVS localized at peroxisomes (Berg et al., 2012). When peroxisome
biogenesis is defective, the mitochondria also exhibit a dysfunction (Baumgart et al.,
2001). For this reason, when defect AKAP1 causes mitochondrial dysfunction,
repercussions might also affect peroxisomes.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated
transcription factors. PPARs and their heterodimeric partner (retinoid X receptor, RXR)
regulate target gene expression by binding the peroxisome proliferator responsive
elements (PPREs) present in its promoter. Lipid metabolism is under the control of
PPARs and PGC-1α (PPARγ coactivator-1α) in both peroxisomes and mitochondria
(Colasante et al., 2015; Finck, 2007). For example, PPARs regulated the transcripts
level of the acyl-CoA oxidase 1 (ACOX1) in peroxisomal β-oxidation and carnitinepalmitoyltransferase (CPT1), acyl-CoA dehydrogenease (ACDH) and 3-ketoacylcoenzyme A thiolase (THIO) in mitochondrial β-oxidation (Colasante et al., 2015).
PPARs also play an important role in β-oxidation of fatty acids in testis, which
associated with testicular functions (steroids synthesis). This subfamily consists of 3
isotypes: PPARα, -β and -γ. PPARα was known to be upregulated by FSH in the testis.
However, PPARγ/RXRγ complex is dominantly expressed in mature Sertoli cells
(Thomas et al., 2010). In addition, PPARγ regulates the lipolytic response by
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influencing the mRNA level of PKAcatα, Akap1, lipase gene Pnplaz (ATGL) and Lipe
(HSL). It was showed that PKAcatα and Akap1 mRNA were downregulated in adipose
tissues of humans. Moreover, a luciferase assay confirmed this PPARγ regulation
mechanism by identifying PPRE motifs at -600bp in the PKAcatα promoter (5’CGACCCTTGACCC-3’)
and
at
>7kb
in
the
Akap1
promotor
(5’-
AGGGAAGAGGTCA-3’) (Rodriguez-Cuenca et al., 2012). In the testis, lipids are not
only acting as energy source, but also as composition of spermatozoa membrane, such
as long-chain fatty acids. Therefore, it is important to study male fertility by making a
further clarification on the role of PPARγ-AKAP1 in lipid metabolism between
mitochondria and peroxisome in the Sertoli cell.
5.7 The role of AKAP1-StAR interaction in Sertoli cells
It is known that AKAP1 anchors PKA to the mitochondrial outer membrane to
phosphorylate the StAR protein. Phosphorylated StAR transports cholesterol from the
outer to the inner mitochondrial membrane, thereafter the cholesterol is converted by
P450scc (cholesterol side-chain cleavage enzyme, also called CYP11A1) to
pregnenolone, which is the rate-limiting step in steroidogenesis (Castillo et al., 2014).
This bioprocess occurs under the stimulation of LH in the theca of interstitial ovary
cells in the female and Leydig cells of the testis in the male (Figure 57 upper portion).
However, there are limited studies on the FSH and StAR expression in Sertoli cells.
One study in 1998 reported that the regulation of FSH to StAR was not associated with
cholesterol delivery and pregnenolone production but was rather associated with free
fatty acid import into mitochondria (Gregory and DePhilip, 1998). Here we showed that
the Akap1 and Star mRNAs are expressed in the TM4 Sertoli cell line and that the
overexpressed AKAP1 protein is localized in mitochondria in these cells under standard
cell culture conditions for TM4 cells. We were also able to confirm that FSH
stimulation resulted in elevated StAR protein amounts in TM4 Sertoli cells (Figure 4344&51). To get a better understanding on steroidogenesis regulation, it would be
interesting to explore further questions, such as: a) the role of AKAP1 and StAR in
mitochondria in Sertoli cells, b) whether AKAP1 and StAR are also expressed in
granulosa cell in the ovary of female (Figure 57 lower portion). Due to time restrictions,
we were not able to test out any other experimental conditions, such as lipid treatment,
hypoxia or other conditions that might provoke an additional targeting of AKAP1 to
peroxisomes.
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Figure 57 Testis steroidogenesis pathway cooperated between Leydig cell and Sertoli cells. This
schematic representative picture includes the integrated information from literatures (Misra et al., 2002;
Wahli et al., 1995; Walker and Cheng, 2005), KEGG Pathway Database① and findings and hypothesis
from this thesis.
①
①
http://www.kegg.jp/keggbin/highlight_pathway?scale=1.0&map=map04913&keyword=FSH%20LH%20StAR
Page | 130
5.8 AKAP4 detection and in vitro localization
AKAP4 plays a conserved role in spermatogenesis throughout mammalian
evolution. A study on Akap4 gene characterization in a marsupial showed that, Akap4
was not observed in any stage of the juvenile developing testis, and could only be
detected in adult testis but not in any other adult tissue. The AKAP4 2.9kb mRNA was
present in the cytoplasm of round and elongated spermatids. After the cleavage of the
precursor pro-AKAP4, the mature AKAP4 protein was then transferred and targeted to
the principal piece of the flagellum in the sperm tail, confirming the same observation
by Nipper in 2006 (Hu et al., 2009; Nipper et al., 2006).
Unfortunately, a good model system of germ cell lines to study spermatogenesis
up to spermatozoa development is not available (Lüers et al., 2006). Only GC2 cell is an
available spermatocyte cell line, however the cells died early after transfection
experiments (non-published data of our group). Also other groups experienced this
problem and had used somatic cell lines (NIH3T3 and HEK293) for overexpression
studies of an AKAP4-GFP function protein. A series of AKAP4-GFP fusion
constructions were expressed. The presence of the pro-domain, the GFP-pro-AKAP4
fusion protein displayed a diffuse cytoplasmic localization. The mature GFP-AKAP4
fusion protein showed a punctate cytoplasmic pattern instead. It was shown that the
pattern of fusion protein localization was dependent on neither the type of cell line, nor
the position of GFP (N- or C-termini) (Nipper et al., 2006). So the AKAP4 targeting
signal sequence is probably not located in N- or C-terminus, but was suggested to be
localized in the middle piece (Hu et al., 2009).
Consistent with these observations, Akap4 mRNA was solely detected in mouse
testis tissue in this study (Figure 43), but not in any other organs or TM4 cell line which
was derived from immature mouse Sertoli cells (Figure 44). The expression of either Nterm or C-term myc-tagged full length AKAP4 in TM4 cells proved to be extremely
difficult and seemed to interfere with their viability. We were able to detect only one or
two transfected cells which displayed a cytoplasmic staining in TM4 cells (Figure 5253). In comparison to NIH3T3 and HEK293, transfections of full-length AKAP4 in
TM4 cells seemed to be detrimental for the cell survival.
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6 Outlook (future plan)
Many different external stimulations could initiate the cAMP generation and
PKA activation. To get an appropriate downstream response it is critical to maintain a
healthy and balanced cellular state. AKAPs enhance the efficiency of cAMP signaltransducing pathways by tethering PKA near sites of cAMP generation or PKA targets,
and rigidly control the downstream response through specific spatial and temporal
expression (McConnachie et al., 2006). Further research on the precise functional
residues of AKAP complex and its tertiary and quaternary structure, could be a solution
for gene cure application in medicine. For example, we supposed that the myc-tag in the
C-terminus hindered the spatial structure of the AKAP1 KH domain. Further studies
using overexpression of the recombinant AKAP1 without the KH domain or replaced
with a junk sequence could verify whether it exerts an influence on its localization and
the observed low SOD2 levels in mitochondria. A series of point mutation assays could
provide answers concerning the critical binding residues.
AKAPs form signal transduction units, “transduceosomes”, which include not
only protein kinases and phosphateases, but also adenylate cyclase (AC), PDE and
mRNA. For example, AKAP1 and other binding partners form a “transduceosome”
complex at the OMM that organizes different signaling enzymes, receptors, adaptor
molecules and mRNAs. The “transduceosome” focuses and functionally integrates
distinct transduction pathways, placing the enzymes into specific sub-cellular contexts
and optimizing signaling events on co-localized effectors. This type of regulation, based
on protein modification, can rapidly adapt mitochondrial activity to changes in
metabolic demands and nutrients availability (Carlucci et al., 2008). At present, 3
recombinant AKAPs plasmids have been constructed with both N- and C-terminal myc
tag (Figure 45). Further co-immunoprecipitation assays and construction of deletion
mutants could be used for “transduceosome” research of AKAP1, 4 and 10, in order to
reveal their binding partners and signaling pathway cross-talks.
Taken together, the data on AKAP1, 4 and 10 suggests their important role on
precise regulation of cellular phosphorylation events. Future studies will focus on
AKAPs sequence feature and metabolic pathway regulation mechanism.
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7 Summary
Sertoli cells in the testis regulate spermatogenesis and are involved in the
molecular pathogenesis of male infertility, an increasing problem worldwide. They are
controlled by testosterone and FSH signaling, which increases the intracellular cAMPlevels, leading to the activation of protein kinase A (PKA) and downstream
phosphorylation events. Compartmentalization of PKA-dependent phosphorylation is
controlled by a variety of distinct A-kinase anchoring proteins (AKAPs), tethering PKA
by high affinity binding to a particular subcellular location. In this respect, it is of
interest that rat AKAP220, partially homologous to the AKAP11 family, was described
in peroxisomes of rat Sertoli cells, essential organelles for maintenance of normal lipid
homeostasis and spermatogenesis regulation in this cell type.
Therefore, the aims of this thesis were to analyze the effects of FSH treatment on
peroxisomal gene expression, to characterize the effect of PKA-AKAP220 signaling on
peroxisomes and to eventually discover new AKAP family members localized in this
cell organelle or in mitochondria in Sertoli cells. Mouse TM4 Sertoli cells were used as
cell culture model system to study the effects of FSH signaling on the peroxisomal and
mitochondrial compartment. Since it is well established in the literature that the
mitochondrial steroidogenic acute regulatory protein (StAR) is stimulated by LHreceptor/PKA-AKAP-signaling in testicular Leydig cells, the Star mRNA expression
was assessed in TM4 Sertoli cells. Indeed, the Star gene was expressed in TM4 Sertoli
cells and its expression level was also FSH-regulated. With optimized cell culture and
FSH-treatment conditions, the gene expression for a variety of peroxisomal biogenesis
and metabolic proteins was analyzed. FSH-treatment induced the expression of Pex11α,
Pex13, Cat, Acox2, and Abcd2 mRNAs, encoding proteins involved in proliferation,
matrix protein import, ROS and lipid metabolism in peroxisomes, demonstrating that
the organelle is influenced by FSH signaling. To verify the peroxisomal localization of
AKAP220 (=AKAP11) and to study its effects on FSH-PKA-mediated spermatogenesis
regulation, this protein was localized with a commercial antibody in different
subcellular fractions and highly purified peroxisomes of TM4 cells. However, our
follow-up experiments with Western blots, bioinformatic evolutionary tree analysis for
conserved PTS1 signals in AKAPs, and sequence alignments between Akap11 cDNA
and genome sequences revealed that the reported cloned rat Akap220 cDNA as well as
the subsequent results of AKAP220 subcellular localization were artifacts.
Page | 133
Furthermore, during the series of experiments on rat Akap11 mRNAs in our
laboratory, total RNA from a variety of rat tissues was isolated and the tissue-specific
Akap11 gene expression was analyzed with specific sets of primer pairs, which revealed
the highest Akap11 mRNA levels in the testis, pituitary, total brain and spleen and
intermediate levels in liver, lung, heart and skeletal muscle. The same primers were
used for subcloning parts of Akap11 in plasmid vectors for in vitro transcription of DIGlabelled cRNA-probes for Akap11 as well as corresponding mRNA controls. A nonradioactive Northern blot method was established, which can be used for future
experiments on alternative splicing of Akap mRNAs.
During the intensive bioinformatics screenings with partially program developed
from Python and R in this study, new AKAP proteins (AKAP1, AKAP4 and AKAP10)
were identified with a putative peroxisomal targeting signal 1 (PTS1), which were also
highly expressed in testis as shown by RT-PCR analysis. Thereafter, series of
subcellular localization experiments were performed to confirm the PTS1 prediction
results. For this purpose, the complete open reading frames of mouse Akap1, Akap4 and
Akap10 cDNAs were cloned into two different myc-tag vectors for expression of N- or
C-terminal myc-tagged fusion proteins in TM4 Sertoli cells. As controls, myc-tagged
GFP constructs were made in the same plasmid vectors, which were used for
optimization of TM4 cells transfection and detection of the expressed fusion proteins by
immunofluorescence with an anti-myc antibody. The expressed N- or C-terminal myctagged fusion proteins of all AKAPs (AKAP1, AKAP4 and AKAP10) were detected
and tried to colocalize with mitochondria or peroxisomes by using doubleimmunfluoresecence with antibodies against myc/SOD2 or myc/PEX14p, followed by
analysis with conventional fluorescence microscopy or confocal laser scanning
microscopy (CLSM). Only AKAP1-C-myc exhibited a typical mitochondrial
distribution pattern and colocalization with SOD2 and exerted strong effects on the
appearance of the mitochondrial network, whereas all other constructs exhibited a
cytoplasmic localization pattern. Effects on SOD2 regulation in mitochondria were also
noted. There was no co-localization of any of the fusion proteins with peroxisomes,
suggesting that the putative C-terminal PTS1 in the AKAPs is not functional. In N-mycAKAP10 preparations, the peroxisomes exhibited a more rod-shaped and less tubular
pattern and giant cells were present. The absence of a single cell with AKAP10-C-myc
expression indicates induction of cell death with yet unknown mechanism.
Page | 134
In conclusion, this thesis established the basic methods and the necessary
recombinant tools were established to analyze the functions of several AKAPs in a
Sertoli cell model system. AKAPs with putative C-terminal targeting signals (AKAP1,
AKAP4, AKAP10) were not targeted to peroxisomes, but exerted strong effects on
mitochondria or cell survival. How these proteins exert these effects on a molecular
basis and whether other AKAP families are involved in the control of peroxisomal
metabolism remains to be elucidated in the future.
Page | 135
8 Zusammenfassung
Sertoli-Zellen im Hoden regulieren die Spermatogenese und wirken an der
molekularen
Pathogenese
männlicher
Infertilität
mit,
einem
zunehmenden
medizinischen Problem weltweit. Sie werden durch Testosteron und den FSHSignalweg kontrolliert, der den Anstieg von intrazellulärem cAMP induziert, zur
Aktivierung
der
Proteinkinase
A
(PKA)
führt
und
nachfolgende
Phosphorylierungsreaktionen auslöst. Die subzelluläre Lokalisierung PKA-abhängiger
Phosphorylierungsprozesse wird durch verschiedene „A-kinase anchoring“-Proteine
durchgeführt, die PKA durch hochaffine Bindung in spezifischen zellulären
Kompartimenten verankern. Hierbei ist von Interesse, dass AKAP220 (=AKAP11) in
Sertoli-Zellen der Ratte in Peroxisomen beschrieben wurde, essentiellen Zellorganellen
in diesem Zelltyp zur Aufrechterhaltung der Homöostase des Lipidstoffwechsels und
der Regulierung der Spermatogenese.
Deshalb waren die Ziele dieser Dissertation, die Auswirkung von FSH auf die
peroxisomale
Signalkaskade
Genexpression
auf
sowie
Peroxisomen
die
Auswirkungen
aufzudecken
und
der
eventuell
PKA-AKAP220neue
AKAP-
Familienmitglieder in dieser Zellorganelle oder in Mitochondrien zu charakterisieren.
Maus TM4 Sertoli-Zellen wurden als Zellkulturmodell zum Studium der FSH-Effekte
auf das peroxisomale und mitochondriale Kompartiment etabliert. Weil aus der
Literatur bekannt ist, dass das „steroidogene akut regulatorische Protein“ (StAR) durch
den LH-receptor/PKA-AKAP-Signalweg in testikulären Leydig-Zellen stimuliert wird,
wurde die Star mRNA-Expression in TM4-Sertoli-Zellen untersucht. Tatsächlich, war
die Star-Gen-Expression in TM4 Sertoli-Zellen vorhanden und auch die FSHvermittelte Regulierung des Star-mRNA-Spiegels konnte nachgewiesen werden. Nach
Optimierung der Zellkulturbedingungen und der FSH-Konzentrationen, wurde die
Expression einer großen Anzahl von Genen für Biogenese- und metabolische Proteine
der Peroxisomen analysiert. FSH-Behandlung führte zur erhöhten Expression von
Pex11α-, Pex13-, Cat-, Acox2- und Abcd2-mRNAs, die Proteine für Proliferation,
Matrixproteinimport, ROS-und Lipid-Stoffwechsel in Peroxisomen kodieren, was die
Beeinflussung dieser Zellorganelle durch den FSH-Signalweg demonstriert. Um die
peroxisomale Lokalisation von AKAP220 nachzuweisen und dessen Effekt auf die
FSH-/PKA-vermittelte Spermatogeneseregulierung auszutesten, wurde versucht, dieses
Page | 136
Protein mittels eines gekauften Antikörpers in unterschiedlichen subzellulären
Fraktionen
und
erbrachten
alle
hochgereinigten
Peroxisomenfraktionen
Folgeexperimente
mit
Western
nachzuweisen.
blots,
Jedoch
bioinformatischer
Evolutionsanalyse von AKAP-Proteinen mit konservierten PTS1-Sigalen sowie
Sequenzvergleiche
zwischen
Akap11-cDNA
und
-Genomsequenzen, dass
die
beschriebene, klonierte Ratte Akap220-cDNA sowie auch alle nachfolgenden Resultate
über die subzelluläre Lokalisation des AKAP220-Proteins, Artefakte in der publizierten
Literatur darstellten.
Innerhalb der
Serie von Experimenten zur Akap11-mRNA-Expression in
unserem Labor, wurden eine große Anzahl von Gesamt-RNA-Proben aus verschiedenen
Rattenorganen isoliert und die gewebsspezifische Expressionsanalyse der Akap11mRNA unter Verwendung spezifischer Primerpaare analysiert, welche die höchsten
Akap11-mRNA-Spiegel in Gesamt-RNA aus Hoden, Hypophyse, Gehirn (gesamt) und
Milz erbrachte, gefolgt von mittelhohen Mengen in Leber, Lunge, Herz und
Skelettmuskel. Mithilfe der gleichen Primerpaare wurde die Subklonierung von
verschiedenen Akap11-Anteilen in Plasmidvektoren zur Herstellung von DIGmarkierten cRNA-Proben für Akap11 durchgeführt, entsprechende Vektoren für interne
Kontrollen kloniert und eine nichtradioaktive Northern Blot-Methode etabliert, mit
deren Hilfe in zukünftigen Experimenten alternative Spleißformen von Akap-mRNAs
nachgewiesen werden können.
Während der intensiven bioinformatischen Analysen mit teilweise selbst
geschriebenen Softwareprogrammen, wurden neue AKAP-Proteine (AKAP1, AKAP4,
AKAP10) mit potentiellen peroxisomalen Zielerkennungssignalen (PTS1, „peroxiosmal
targeting signal 1“) identifiziert, die auch in hoher Menge im Hoden vorkommen, wie
durch RT-PCR-Analyse gezeigt wurde. Um die bioinformatische PTS1-Vorhersage zu
verifizieren, wurden im Folgenden Serien von Experimenten zur subzellulären
Lokalisation dieser Proteine durchgeführt. Für diesen Zweck wurden jeweils die
proteinkodierenden Regionen PCR-amplifizierter Akap1-, Akap4- und Akap10 –cDNAs
der Maus in zwei unterschiedliche myc-Tag-Vektoren zur Expression N- und Cterminaler myc-Fusionsproteine in TM4 Sertoli-Zellen kloniert. Als Kontrollvektoren
wurden myc-Tag-GFP-Konstrukte der gleichen Vektoren hergestellt, die die
Optimierung der TM4 Sertol-Zell-Transfektion und der Detektion der mycFusionsproteine durch Immunfluoreszenz mittels eines
anti-myc-Antikörpers
Page | 137
ermöglichten.
Die exprimierten N- und C-terminalen myc-Fusionsproteine aller
AKAPs (AKAP1, AKAP4, AKAP10) wurden durch Doppelimmunfluoreszenz mit
Antikörpern gegen myc/SOD2 oder myc/PEX14p detektiert und versucht, mit
Mitochondrien oder Peroxisomen zu kolokalisieren. Alle DoppelimmunfluoreszenzPräparate wurden mittels konventioneller Fluoreszenzmikroskopie und konfokaler
Laserscanning-Mikroskopie (CLSM) ausgewertet. Nur das exprimierte AKAP1-C-mycProtein zeigte ein typisch mitochondriales Verteilungsmuster, kolokalisierte mit SOD2
und induzierte starke Veränderungen des mitochondrialen Netzwerks, während alle
anderen exprimierten Fusionsproteine eine cytoplasmatische Lokalisation aufwiesen.
Kolokalisation der exprimierten Fusionsproteine mit Peroxisomen wurde nicht
gefunden, was für die fehlende Funktionalität der putativen PTS1-Sequenz am Cterminalen Ende der AKAPs für den Import in Peroxisomen spricht. Jedoch wies das
morphologische
exprimierenden
Organellenmuster
Zellen
vermehrt
der
runde
Peroxisomen
und
weniger
in
N-myc-AKAP10-
tubuläre
Formen
auf.
Interessanterweise wurde in parallelen Experimenten keine einzige Zelle mit AKAP10C-myc-Expression nachgewiesen, was für die Induktion von Zelltod durch dieses
Fusionsprotein mittels eines bisher unbekannten Mechanismus spricht.
Zusammenfassend lässt sich feststellen, dass in dieser Dissertation die
grundlegende Methodik und die notwendigen rekombinanten Werkzeuge zur Analyse
der Funktionen verschiedener AKAPs in einem in einem Sertoli-Zellkultur-Modell
etabliert wurden. AKAPs mit putativen PTS1-Sequenzen (AKAP1, AKAP4, AKAP10)
wurden nicht in Peroxisomen importiert, lösten jedoch starke Effekte in dem
mitochondrialen Netzwerk und auf das Zellüberleben aus. Wie diese Proteine diese
Effekte auf molekularer Ebene auslösen und ob andere AKAP-Familien in die Kontrolle
des peroxisomalen Metabolismus eingeschaltet sind, soll in der Zukunft aufgeklärt
werden.
Page | 138
9 List of abbreviations
ABC
ATP-binding cassette
ABP
Androgen-binding protein
AC
Adenylyl cyclase
ACDH
Acyl-CoA dehydrogenease
ACOX1
Acyl-CoA oxidase 1
ADAPS
Alkyl dihydroxyacetonephosphate synthase
AKAP
A-kinase anchoring protein
Ago2
Argonaute 2
AGT1
Glyoxylate aminotransferase 1
ALD/AMN disease
Adrenoleukodystrophy/Adrenomyeloneuropathy disease
Amp
Ampicillin
AR
Androgen receptor
ARE
Androgen response elements
ARTs
Assisted reproductive techniques
AQP2
Aquaporin 2
AVP
Arginine vasopressin
BTB
Blood-testis barrier
BSA
Bovine Serum Albumin Acetylated
Ca2+
Calcium
CaM
Calmodulin
cAMP
Cyclic adenosine monophosphate
CaN (PP2B)
Calcium-activated Ser/Thr phophoatase calcineurin
CCS
Copper chaperone of SOD1
CLSM
Confocal laser scanning microscopy
CNG (cNGC)
Cyclic nucleotide gated ion channel
COX
Cytochrome c oxidase
CPT1
Carnitine-palmitoyltransferase
CREB
cAMP response element binding protein
CT
Cholera toxin
DAPAT
Dihydroxyacetonephosphate acyltransferase
D/D domain
Dimerization/docking domain
DEPC
Diethyl pyrocarbonate
Drp1
Dynamin related protein 1
Page | 139
DIG
Digoxigenin
eSpt
elongated spermatids
Siah2
E3 ubiquitin-protein ligase 7 in absentia homolog 2
SOD1
Cu/Zn superoxide dismutase 1
EDAs
Endocrine disrupting agents
EGF
Epidermal growth factor
Epac
Exchange proteins directly activated by cAMP (cAMP-activated GTP-exchange factors)
EST
Expressed sequence tag
F0-f
ATP synthase F0 complex
FGF
Fibroblast growth factor
Fis1
Fission factor 1
FSH
Follicle-stimulating hormone
FSHR
Follicle-stimulating hormone receptor
FSIP1
Fibrous Sheath Interacting Proteins 1
FSK
Forskolin
GNPAT
Glyceronephosphate O-acyltransferase
GPCR
G protein-coupled receptor
GSK-3β
Glycogen synthase kinase 3β
GV stage
Germinal vesicle stage
HGNC
Human genome organization nomenclature committee
HIF-1α
Hypoxia induced factor 1α
HIV-1 RT
HIV-1 reverse transcriptase
IBMX
3-Isobutyl-1-methylxanthine
IGF-1
Insulin-like growth factor-1
IMAGE
Intergrated Molecular Analysis of Gonomes and their Expression
IQGAP1
IQ domain GTPase-activating protein 1
IVF
in vitro fertilization
KH domain
K homology domain
LDHA
Lactate dehydrogenase A
LDHB
Lactate dehydrogenase B
LPL
Lipoprotein lipase
LH
Luteinizing hormone
LHR
Luteinizing hormone receptor
MAVS
Mitochondrial antiviral signaling adaptor
Mff
Mitochondrial fission factor
Mfn1
Mitofusion1
Page | 140
MFP-2 (HSD17B4)
Multifunctional protein-2
MIS
Mullerian-inhibiting substance
MnSOD (SOD2)
Manganese superoxide dismutase
MOM
Mitochondrial outer membrane
MVBs
Multivesicular bodies
MYCBP (AMY-1)
c-myc binding protein
NMDA
N-methyl-D-aspartate receptor 2B
NMJ
Neuromuscular junction
ODFs
Outer dense fibers
OPA1
Optic atrophy 1
OPLs
Oral premalignant lesions
P450scc
(CYP11A1)
cytochrome P450 side-chain cleavage enzyme
PBS
Phosphate-buffered saline
PC
Prostate cancer
PCR
Polymerase-chain reaction
PDE
Phosphodiesterase
PGC-1α
PPARγ coactivator -1α
PKA
Protein kinase A
PMD1
Peroxisomal and Mitochondrial Division Factor 1
POMA
Paraneoplastic opsoclonus-myoclonus-ataxia
PP1
Protein phosphatase 1
PPARγ
Peroxisome proliferator-activated receptor γ
PPRE
Peroxisome proliferator responsive element
PTC
Peritubular cells
PTPD1
Protein tyrosine phosphatase D1
PTS1
Peroxisomal targeting signal 1
PTSP
Peroxisomal Target Signal Predictor
PUFA
Polyunsaturated fatty acids
PVDF
Polyvinylidene fluoride
RIAD
RI-anchoring disruptor
RIIBD
RII-binding domain
RB protein
Retinoblastoma protein
rSpt
round spermatids
RXR
Retinoid X receptor
SC
Sertoli cells
Page | 141
SCD
Sudden cardiac death
SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Siah2
Seven in absentia homolog
Spc
Spermatocytes
Spg
Spermatogonia
StAR
Steroidogenic acute regulatory protein
TDS
Testicular Dysgenesis Syndrome
THIO
3-ketoacyl-coenzyme A thiolase
TSHR
Thyroid-stimulating hormone receptor
TGF
Transforming growth factor
TSHR
Thyroid-stimulating hormone receptor
3’-UTR
3’ untranslated region
V2R
Vasopressin type 2 receptor
VLCFA
Very long chain fatty acids
WHO
world health organization
Page | 142
10 Figure index
Figure 1 Schematic illustration of testis structure (Figure 1A) and the hypothalamicpituitary-testicular axis (Figure 1B). ......................................................................... 9
Figure 2 FSH stimulated cAMP-PKA signaling pathway (Figure 2A) and localization
of PKA Cα in different cell types of a seminiferous tubule in the testis of a 120dold wild-type mouse (Figure 2B1) and in TM4 Sertoli cells (Figure 2B2-2B3)....... 11
Figure 3 AKAPs assemble the key enzymes and factors ............................................... 15
Figure 4 Alignment of the PKA RII-binding regions of the indicated AKAPs and
artificial disruptor peptides derived......................................................................... 16
Figure 5 Crystal structure (PDB ID: 2HWN) ................................................................. 17
Figure 6 Schematic diagram of hAKAP220................................................................... 19
Figure 7 Schematic diagram of AKAP220 complex leading function in cell migration21
Figure 8 Schematic diagram of mouse AKAP1 (AKAP121)......................................... 23
Figure 9 Structure of a KH domain (PDB ID: 2ANR). .................................................. 23
Figure 10 A model illustrating post-transcriptional and post-translational regulation of
StAR. ....................................................................................................................... 25
Figure 11 A model illustrating of AKAP1 scaffold complex on the mitochondrial outer
membrane ................................................................................................................ 27
Figure 12 Normal sperm structure.................................................................................. 28
Figure 13 Brief overview of major metabolic pathways and peroxins (in number) in
peroxisomes of the mammalian testis. This picture is adapted from (Colasante et
al., 2015; Fahimi and Baumgart, 1999). .................................................................. 33
Figure 14 Schematic illustration of peroxisome purification by OptiPrep™ (keep all
solutions and carry out all operations at 4°C). ........................................................ 57
Figure 15 An example of RNA-Chip results to check the quality of isolated RNAs..... 60
Figure 16 workflow of Northern blot ............................................................................. 61
Figure 17 Phylogenetic tree of AKAPs with putative PTS1 .......................................... 75
Figure 18 Conventional RT-PCR of internal control and positive control .................... 78
Page | 143
Figure 19 Conventional RT-PCR results of alterations of mRNA levels of peroxins on
peroxisome proliferation ......................................................................................... 78
Figure 20 Conventional RT-PCR results of alterations of mRNA levels of peroxins on
peroxisomal matrix protein import.......................................................................... 78
Figure 21 Conventional RT-PCR results of alterations of mRNA levels of antioxidative
enzymes ................................................................................................................... 79
Figure 22 Conventional RT-PCR results of alterations of mRNA levels of Acyl-CoA
oxidases ................................................................................................................... 79
Figure 23 Conventional RT-PCR results of alterations of mRNA levels of fatty acid
transporters .............................................................................................................. 80
Figure 24 Conventional RT-PCR results of PPAR transcription factors ....................... 80
Figure 25 Peroxisome marker PEX14p .......................................................................... 82
Figure 26 Western blot of AKAP220 ............................................................................. 82
Figure 27 BLAST Results of Akap11 mRNAs from Genome Database ....................... 85
Figure 28 The sequence in the orange frame shows two potential EcoRI linkers in
mRNA [U48288.1]. ................................................................................................. 87
Figure 29 Schematic illustration of the alignment and position of Akap11 rat mRNA . 89
Figure 30 Constructed plasmids maps, including Akap11 rat mRNA specific
hybridization probes LSP (230bp), BSP1 (308bp), BSP2 (893bp) and BSP3
(608bp) and housekeeping gene probes, such as GAPDH (417bp), β-actin (120bp),
28S rRNA (254bp), and StAR (292bp). .................................................................. 90
Figure 31 Plasmids linearization for in vitro transcription............................................. 91
Figure 32 Sensitivity of DIG-labeled antisense RNA probes were verified by dot blot
assay (40s exposure)................................................................................................ 91
Figure 33 Test of 9 different types of washing buffer with 2 distinct dilutions of antiDIG concentration (40s exposure) .......................................................................... 92
Figure 34 Rat tissue total RNA dot blot hybridization with 4 probes ............................ 94
Figure 35 Total RNA separation by electrophoresis ...................................................... 95
Figure 36 Distribution profile of Akap11 mRNA in different rat organs. ...................... 96
Page | 144
Figure 37 PCR product with T7 promotor ..................................................................... 97
Figure 38 PTS1 prediction of all AKAP1 family members in different species ............ 99
Figure 39 PTS1 prediction of all AKAP4 family members in different species, ......... 100
Figure 40 PTS1 prediction of all AKAP10 family members in different species, ....... 101
Figure 41 Akap1, Akap4 and Akap10 distribution profile ............................................ 102
Figure 42 Akap1, Akap4 and Akap10 mRNA are high expressed in testis. ................. 103
Figure 43 Test of designed primers for Akap1, Akap4, Akap10, and their expression
level in different mouse organs ............................................................................. 104
Figure 44 Test expression of Akap1, Akap4, Akap10 in TM4 Sertoli cell line ............ 105
Figure 45 The instruction of myc-tag recombined plasmids generation (inserts are:
GFP, AKAP1, AKAP4 and AKAP10) .................................................................. 108
Figure 46 Co-localization test for the myc-tagged GFP-fusion proteins as positive
controls, N-terminal myc-labelled GFP (GFP-3B) plasmid and C-terminal myclabelled GFP (GFP-5B) plasmid. .......................................................................... 110
Figure 47 Immunofluorescence images showing peroxisomal membrane staining (A-D)
in untreated TM4 cells........................................................................................... 111
Figure 48 Immunofluorescence images showing peroxisomal (A-C) and mitochondrial
(D-F) matrix staining in untreated TM4 cells.. .................................................... 112
Figure 49 Double-immunofluorescence showing for the myc-tag and PEX14p,
revealing that both myc-tagged AKAP1 fusion proteins did not co-localize with
peroxisomes........................................................................................................... 113
Figure 50 Double-immunofluorescence showing that N-terminal myc-tagged AKAP1
(AKAP1-3B) was not co-localized with mitochondria (Figure 50A-C), but Cterminal myc-tagged AKAP1 (AKAP1-5B) was (Figure 50D-F).. ...................... 114
Figure 51 CLSM images of double-immunofluorescence for myc and SOD2, showing
that N-terminal myc-tagged AKAP1 (AKAP1-3B) was not co-localized with
mitochondria (Figure 51A-B), but C-terminal myc-tagged AKAP1 (AKAP1-5B)
was (Figure 51C-D).. ............................................................................................. 116
Page | 145
Figure 52 Double-immunofluorescence preparations for myc and SOD2, showing two
types of myc-tagged AKAP4 were both expressed in the cytoplasm and did not colocalize with mitochondria in TM4 cell line.. ....................................................... 117
Figure 53 CLSM images of double immunofluorescence preparations for myc and
SOD2, showing two types of myc-tagged AKAP4 fusion proteins which were both
localized with cytoplasm and did not target to mitochondria in TM4 cell line.. .. 118
Figure 54 Double-immunofluorescence preparation for myc and PEX14p, showing that
N-terminal myc-tagged AKAP10 (AKAP10-3B) did not co-localize with
peroxisomes........................................................................................................... 119
Figure 55 Double-immunofluorescence preparation for myc and SOD2, showing that
the N-terminal myc-tagged AKAP10 (AKAP10-3B) did not co-localize with
mitochondria.......................................................................................................... 119
Figure 56 CLSM images of a double immunofluorescence preparation for myc and
SOD2, showing that the N-terminal myc-tagged AKAP10 (AKAP10-3B) did not
co-localize with mitochondria.. ............................................................................. 120
Figure 57 Testis steroidogenesis pathway cooperated between Leydig cell and Sertoli
cells........................................................................................................................ 130
Page | 146
11 Tabel index
Table 1 Cell line and medium (information from ATCC search and Health Protechtion
Agency cell line data sheet 50004291) ................................................................... 36
Table 2 Routine reagents list .......................................................................................... 36
Table 3 Buffer recipes .................................................................................................... 39
Table 4 Commercial plasmids information .................................................................... 41
Table 5 List of primers designed and used in this study ................................................ 42
Table 6 Antibody dilution list ......................................................................................... 47
Table 7 Properties and preparation of Iodixanol-sucrose solution ................................. 55
Table 8 Taq-PCR reaction .............................................................................................. 65
Table 9 Pfu-PCR reaction ............................................................................................... 65
Table 10 Restriction enzyme digestion of GFP and 3 AKAPs (4h incubation at 37°C) 67
Table 11 Ligation reaction (overnight incubation at 4°C).............................................. 68
Table 12 Chemical structure of commen used antibiotics .............................................. 70
Table 13 AKAP1-Mutation-PCR reagent....................................................................... 70
Table 14 AKAP1-Mutation-PCR Dpn I digestion (4h incubation at 37°C) ................... 71
Table 15 Summarized PTS1 tripeptides derived from the “Peroxisomal Target Signal
Predictor (PTSP)”.................................................................................................... 74
Table 16 Optimization of cell density to obtain a monolayer of TM4 cells on Day 4-6 77
Table 17 Total RNA was isolated using combined methods, and the RNA quality was
checked by RNA-Chip analysis .............................................................................. 87
Table 18 Total RNA precipitation .................................................................................. 97
Page | 147
12 Supplemental data
S 1 Python codes used in this study (see subsection 3.1.3)
seq_num_len_des_read-code.py:
from Bio import SeqIO
AKAP_file = 'All AKAP1 protein from NCBI-143 seq.fasta'
Read_file = 'seq_num_len_des_read.txt'
Handle = open(Read_file,'w')
Handle.write("seq_index\t" + "seq_length\t" + "seq_description\n")
num = 0
for seq_record in SeqIO.parse(AKAP_file,'fasta'):
num = num + 1
seq_len = str(len(seq_record.seq))
seq_des = str(seq_record.description)
Handle.write(str(num) + ':\t' + seq_len + 'aa\t' + seq_des + '\n')
Handle.close()
Remove_duplicate_seq-code.py:
from Bio.Seq import Seq
from Bio.SeqRecord import SeqRecord
seq_map_des = {}
AKAP_file = 'All AKAP1 protein from NCBI-143 seq.fasta'
Handle_out = open("seq_without_duplicate.txt", "w")
Handle_id = open("id_order.txt", "w")
for seq_record in SeqIO.parse(AKAP_file, "fasta"):
seq_str = str(seq_record.seq)
seq_des = str(seq_record.description)
if seq_str not in seq_map_des:
seq_map_des[seq_str] = [seq_des]
else:
if len(seq_des) > len(seq_map_des[seq_str][0]):
seq_map_des[seq_str].insert(0,seq_des)
else:
seq_map_des[seq_str].append(seq_des)
num = 0
for map_seq in seq_map_des:
Handle_out.write(">" + seq_map_des[map_seq][0] + "\n" + map_seq + "\n")
num = num + 1
Handle_id.write(str(num) + ":\t" + str(len(map_seq)) + "aa\t" + ' '.join(seq_map_des[map_seq]) + '\n')
Handle_out.close()
Handle_id.close()
AKAP_domain_selection-code.py:
import re
AKAP_file = 'seq_without_duplicate.txt'
True_file = 'AKAP_true_protein.txt'
False_file = 'AKAP_false_protein.txt'
True_file_info = 'AKAP_true_protein_info.txt'
Page | 148
Handle_info = open(True_file_info, 'w')
Handle_info.write('Seq_num' + '\t' + 'Seq_ID' + '\t' + 'Length_aa' + '\t' + 'AKAP_domain' + '\t' +
'AKAP_Start' + '\t' + 'AKAP_End' + '\n')
Handle_False = open(False_file,'w')
Handle_True = open(True_file,'w')
num = 0
for seq_record in SeqIO.parse(AKAP_file,'fasta'):
seq_id = seq_record.id
seq_des = seq_record.description
seq_len = str(len(seq_record))
seq_str = str(seq_record.seq)
pattern = '[AVLISE]..[AVLIF][AVLI]..[AVLI][AVLIF]..[AVLISE]'
find_pattern = re.search(pattern,str(seq_record.seq))
if find_pattern:
num = num + 1
start_site = str(find_pattern.start() + 1)
end_site = str(find_pattern.end())
Handle_info.write(str(num) + '\t' + seq_id + '\t' + seq_len + '\t' + find_pattern.group() + '\t' +
start_site + '\t' + end_site + '\n')
Handle_True.write('>' + seq_des + '\n' + seq_str + '\n')
else:
Handle_False.write('>' + seq_des + '\n' + seq_str + '\n')
Handle_info.close()
Handle_True.close()
Handle_False.close()
PTS1_predictor_filter-code.py:
import urllib
import re
handle_file = open("PTS1_pred_score.txt", "w")
handle_file.write("Seq_num" + "\t" + "Seq_name" + "\t" + "C-terminus_12seq" + "\t" + "Pred_Score"
+"\t" + "Classification" + "\n")
url = 'http://mendel.imp.ac.at/jspcgi/cgi-bin/pts1/pts1.cgi'
num = 0
for seq_record in SeqIO.parse('AKAP_true_protein.txt','fasta'):
num = num + 1
speices_pattern = re.compile("\[(.+)\]")
name_data = "AKAP1_" + speices_pattern.findall(seq_record.description)[0]
pred_in = re.search("PREDICTED", seq_record.description)
if pred_in:
name_data = name_data + "_pred_seq"
else:
name_data = name_data + "_real_seq"
seq_data = str(seq_record.seq)[-20:] + '\n'
query_data = {"function": "3", "name" : name_data, "sequence" : seq_data}
result_info = urllib.urlopen(url, urllib.urlencode(query_data))
result = result_info.read()
C_ter_pattern = re.compile('<tr><td>C-terminus</td><td>(.+)</td></tr>')
score_pattern = re.compile("<tr><td>Score<td>(.+)</td></tr>")
pred_clas_pattern = re.compile("<tr><td>Prediction classification</td><td>(.+)</td></tr>")
Page | 149
C_terminus = C_ter_pattern.findall(result)[0]
score = score_pattern.findall(result)[0]
pred_clas = pred_clas_pattern.findall(result)[0]
handle_file.write(str(num) + "\t" + seq_record.id + "_" + name_data + "\t" + C_terminus + "\t" + score
+ "\t" + pred_clas + "\n")
handle_file.close()
Barplot_AKAP1.R:
Rdata <- read.table("AKAP_PTS1_for_R.txt", sep = "\t", header = TRUE)
domain_start <- Rdata$AKAP_Start
domain_end <- Rdata$AKAP_End
seq_length <- Rdata$Length_aa
seq_name <- as.matrix(Rdata$Seq_name)
PTS1_Pred <- as.matrix(Rdata$Classification)
c_terminus <- as.matrix(Rdata$C.terminus_12seq)
barplot_data = NULL
for(i in 1:length(domain_start))
{
if(PTS1_Pred[i] == "Targeted")
{
barplot_data <- cbind(barplot_data, c(domain_start[i] - 0, domain_end[i] - domain_start[i],
seq_length[i] - domain_end[i], 12))
}
else
{
barplot_data <- cbind(barplot_data, c(domain_start[i] - 0, domain_end[i] - domain_start[i],
seq_length[i] - domain_end[i], 0))
}
}
colnames(barplot_data) = seq_name
png(filename = "AKAP1_domain_feature.png", width = 1200, height = 600, pointsize = 12)
par(mar=c(2, 18, 1, 1),bg = grey(1), ps= 6.5,cex =1)
b <- barplot(barplot_data, horiz = TRUE, col = c("light blue", "green", "light blue", "red"),space = 0.95,
las=1, xlim = c(0, max(seq_length) + 500))
text(x = seq_length + 90, y = b, labels = c_terminus)
dev.off()
pdf(file = "AKAP1_domain_feature.pdf", paper = "a4", width = 12, height = 12)
par(mar=c(2, 18, 1, 1),bg = grey(1), ps= 6.5,cex =1)
b <- barplot(barplot_data, horiz = TRUE, col = c("light blue", "green", "light blue", "red"),space = 0.95,
las=1, xlim = c(0, max(seq_length) + 500))
text(x = seq_length + 180, y = b, labels = c_terminus)
dev.off()
Page | 150
S 2 RNA Chip result to show the quality of total RNA isolated from rat tissues (see subsection 4.5.3)
Page | 151
S 3 The 531 types of C-terminal tripeptides of all known AKAPs retrieved from “UniProt” database (the
organge marked are hits by the Combination Matrix from Table 15)
No.
1
AAA
DEA
ENS
GQE
KEK
LGQ
NIR
PSS
RRR
TES
VLC
2
AAE
DED
ENT
GRD
KER
LIE
NKK
PST
RSR
TEY
VNL
3
ACQ
DEG
EPE
GSR
KGS
LIK
NLF
PTE
RTP
TFD
VQI
4
AEH
DEN
EPL
GTK
KIH
LIL
NLI
PTG
RVK
TFS
VRG
5
AES
DEV
EPT
GVA
KIT
LKF
NLT
PVH
RVL
TGS
VRR
6
AFI
DFS
EPV
HDT
KIV
LKK
NLY
PWN
RVV
THA
VSK
7
AFL
DGF
EQE
HFD
KKA
LKR
NNE
QAC
SAS
THH
VSP
8
AFW
DGK
EQN
HFY
KKI
LKV
NNS
QAD
SCH
TKG
VTE
9
AGP
DGS
ERG
HGK
KKK
LKW
NPG
QAL
SCL
TKI
VTV
10
AGS
DGT
ERH
HLG
KKR
LLH
NPR
QAQ
SDS
TKL
VVE
11
AHQ
DHS
ETL
HLL
KLA
LLK
NPS
QAV
SEA
TKR
VVM
12
AIE
DKK
EVI
HLS
KLC
LLQ
NPY
QDN
SEE
TKV
VYE
13
AIQ
DLI
EVV
HTA
KLL
LLS
NRK
QDY
SEI
TLN
VYS
14
AKG
DMY
FEC
HTE
KLS
LMY
NRR
QEV
SEN
TLT
WEA
15
AKL
DNE
FEM
HTG
KLV
LNA
NRV
QLE
SET
TLV
WLE
16
AKP
DNV
FGT
ICL
KMC
LNF
NSE
QLK
SFL
TMR
WNR
17
AKS
DNY
FHF
IDF
KMF
LQA
NTI
QMF
SFS
TMT
WSG
18
AKV
DQL
FIK
IDL
KMR
LQR
NVE
QNL
SGA
TNL
WSR
19
ALA
DRI
FKK
IEK
KNI
LRR
NVN
QRA
SGC
TPE
WSS
20
ALK
DSF
FLF
IFA
KPE
LRS
NVT
QRR
SGR
TQR
WTA
21
ALP
DSL
FLI
IFC
KPP
LSC
NYK
QRT
SIC
TRR
YAG
22
ALW
DSY
FNL
IFD
KQA
LSE
NYR
QRY
SIS
TSL
YAL
23
ANL
DTQ
FNN
IGA
KQD
LSI
PAC
QSC
SKK
TTC
YAP
24
ARK
DVE
FPE
IGE
KQI
LSK
PAE
QSF
SLE
TTI
YDP
25
ASE
EAE
FSL
IGK
KQN
LSM
PAV
QSG
SLG
TTL
YDT
26
ASG
EAP
FTL
IGR
KQP
LSR
PDP
QSP
SLS
TTV
YGS
27
ASL
EDA
FVK
IIC
KQS
LSV
PDT
QSQ
SNI
TTY
YIW
28
ASP
EDC
FYD
IIL
KRE
LSY
PEE
QTD
SPM
TVM
YKQ
29
ATE
EDN
GAP
IKE
KRL
LTK
PER
QTE
SPR
TVS
YMG
30
ATP
EDS
GCG
ILK
KRN
LTS
PES
QTG
SPS
TVV
YRN
31
ATV
EEE
GDE
IME
KSK
LVL
PFE
QVD
SRA
TYD
YSC
32
AVL
EEG
GEG
INF
KSN
LVQ
PGC
QVL
SRG
TYI
33
AVQ
EEL
GEP
INR
KSP
LVS
PGE
RAA
SRS
TYK
34
AWC
EEN
GET
INT
KTE
MAQ
PGG
RAE
SRV
VAA
35
AYG
EEV
GGA
IPF
KVI
MEE
PGH
RAT
SSG
VAH
36
CCP
EGF
GGG
IPS
KVL
MEQ
PGQ
RGF
SSL
VAP
37
CDI
EGK
GGP
IQE
KVR
MFI
PGS
RGL
SSS
VDS
38
CGK
EGT
GGS
IRR
KVV
MHR
PGW
RGR
SST
VEK
39
CLF
EHA
GIS
ISK
KYL
MHS
PHK
RHL
STE
VER
40
CLI
EIA
GKS
ISN
LDM
MIK
PIL
RKF
STI
VES
41
CMK
EKD
GLG
ITF
LDQ
MKL
PKE
RKI
STK
VEV
42
CPF
EKI
GLH
ITL
LEC
MKQ
PKN
RKY
STL
VFV
43
CRL
EKK
GLP
IYE
LEE
MLE
PLN
RLA
STV
VGH
Page | 152
44
CSK
EKT
GLS
45
46
KAL
LEF
MLV
PLP
CSQ
ELE
CTE
ELG
GME
KCY
GNA
KDQ
47
DAS
ELT
GNE
48
DDD
ENA
RLC
SVL
VHA
LEK
MRR
PLQ
RLE
SVS
VHR
LET
MVV
PLS
RLQ
TAS
VHV
KDS
LFF
NCK
PNV
RMA
TCE
VIV
GNG
KEA
LFL
NDN
PPE
RPV
TEE
VKL
49
DDE
ENI
GNL
KEE
LFQ
NEN
PPS
RQM
TEL
VKT
50
DDQ
ENP
GPA
KEF
LGK
NFS
PRE
RRD
TEM
VLA
S 4 All known PTS1 binding sites downloaded from PTS1 BLOCK (http://216.92.14.62/diy_PTS1.html)
at the “PeroxisomalDB Home” website
No. Peroxisomal proteins
Their last 12-aa residues
Their last 3-aa residues
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
GOX1
GGT1
EHHADH
mmSerhl
mmCrot
AAT2
AT5G18100.1
mmEphx2
OPR3
mmEhhadh
CIT2
DHRS4
LYS4
AOAT2
mmCrat
mmMlycd
HMGCL
NUDT19
MLS
mmAgxt
PTE2b
PTE2
BAAT
GNPAT
DAMOX
mmPte2a
mmGnpat
SPS19
PAOX
EPHX2
CRAT
GSTK1
PRDX5
ACH2
TEWDTPRPSARL
TQYDNNFGYSKM
WQSLAGSPSSKL
LQGLQRMTSARL
HDMIQLMNTAHL
EVVRFYTIEAKL
IIGLQSSADAKL
TEVQNPSVTSKI
TDYPFLAPFSRL
WQSLAGPHSSKL
YKELVKNIESKL
TVVVGGGTPSRL
KGGLEGWVKSQL
SQYADNFGYSRM
RTLLQNHPRAKL
SLVAQFQNNSKL
TSSKVAQATCKL
NYIVNKRYTAHL
IVAHYPINVSRL
REALQHCPKNKL
LGGTQKTAVPKL
LGGHEGTIPSKV
RKHLIPDVTSQL
KTPIGKPATAKL
EKKLSRMPPSHL
LDGKKKTIPAKL
KKPIGKPATAKL
PEALIKSMTSKL
WAPQVQQPRPRL
SDARNPPVVSKM
RALLQSHPRAKL
MGPIPPAVNARL
TCSLAPNIISQL
ARPPKPSGTSKL
ARL
SKM
SKL
ARL
AHL
AKL
AKL
SKI
SRL
SKL
SKL
SRL
SQL
SRM
AKL
SKL
CKL
AHL
SRL
NKL
PKL
SKV
SQL
AKL
SHL
AKL
AKL
SKL
PRL
SKM
AKL
ARL
SQL
SKL
Page | 153
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
IDE
PECI
PTE1
ACOX2
mmPaox
MLYCD
GOX2
IDP3
HAO2
AGT
AT3G06860.1
mmDao1
PDCR
AT5G63380.1
CHY1
MLS1
mmHao3
IDH1
PECR
CAT2
DCI1
mmAcox2
ECH1
mmPecr
CROT
mmBC016076
mmPte1
mmAcox1
FOX2
AT1G68010.1
AT3G51840.1
mmUox
PCS60
DASOX
mmAmacr
NPY1
mmHsd17b4
HSD17B4
MDH3
AGXT
mmDecr2
AT2G35690.1
mmPte2b
mmAcox3
ACOX3
TES1
Page | 154
VKPHINFMAAKL
AVVNFLSRKSKL
IRVKPQVSESKL
IRPLLQSWRSKL
WDSQVEQSRPRL
SLVAQFQKNSKL
TEWDTPRHLPRL
SSNEDKKGMCKL
EINRNLVQFSRL
LQHHIPLIPSRI
LSAPVKQASSRL
EKKLSRLPPSHL
GLPDFASFSAKL
TKIAVDGNASKL
RNNLPALGIAKL
STKATPTDLSKL
EISPDLIQFSRL
ENLKIKLAQAKL
KMKETFKEKAKL
ALENENKRKAKL
KQLQEGNRRHKL
IRPLMQSWKPKL
NKELKTVTFSKL
RIKESFKKKAKL
HDMIQLMNSTHL
LSGDKRPSPAKL
IRLKPQVSESKL
YYKHLKPLQSKL
AAVKLSQAKSKL
NSKALGLPVSKL
IASFKPATRSRL
TGTVKRKLPSRL
TFAKSSRNKSKL
HALRTPIPKSNL
RIVESDKLKANL
YKNLRKTSSSHL
KLQMILKDYAKL
KLQMILKDYAKL
KGKSFILDSSKL
RAALQHCPKKKL
QLLEFESFSAKL
PLIKQRFRSAKL
LGSTHSRASCRL
NKSVADRLKSQL
NKSVADRLKSQL
VYGSERDIRAKF
AKL
SKL
SKL
SKL
PRL
SKL
PRL
CKL
SRL
SRI
SRL
SHL
AKL
SKL
AKL
SKL
SRL
AKL
AKL
AKL
HKL
PKL
SKL
AKL
THL
AKL
SKL
SKL
SKL
SKL
SRL
SRL
SKL
SNL
ANL
SHL
AKL
AKL
SKL
KKL
AKL
AKL
CRL
SQL
SQL
AKF
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
mmHao1
ECI1
HAO1
ACOX1
LONP
mmEch1
mm5330420D20Rik
PIPOX
mmNudt7
GPK1
mmPeci
GTO1
ACOX
AMACR
LYS1
CAT
mmIdi1
mmCat
LVRKNPLAVSKI
RQLGSKQRKHRL
LVRKNPLAVSKI
SYKHLKSLQSKL
VKTRPGLLNSKL
KRDTKSITFSKL
HTLRTPASLSKL
ISRFPSLGKAHL
RTFLWRYSLSKL
NKDNDITTDAKI
AIMSFVSRKPKL
VPLGPKPDISRL
FNWVEANAWSSL
KIIESNKVKASL
RHCARVKRSSRL
GSHLAAREKANL
SPFVDHEKIHRL
TGEPVELKTQAL
SKI
HRL
SKI
SKL
SKL
SKL
SKL
AHL
SKL
AKI
PKL
SRL
SSL
ASL
SRL
ANL
HRL
QAL
Page | 155
S 5 PTS1 prediction of AKAP1, -4, -10 family members
Page | 156
Page | 157
Page | 158
13 Acknowledgements
At first, I am really thankful to Professor Eveline Baumgart-Vogt and the China
Scholarship Council (CSC) committee to give me the opportunity to broad my horizons
in scientific knowledge and international culture.
Since 2011 autumn, I have studied in Justus-Liebig-University Giessen for 4
years. During these 4 years, I have attended lots of seminars and workshops from
International Giessen Graduate Centre for the Life Sciences (GGL), enjoying the
fantastic research from experts and developing more skills. Thanks to the work and
offer from GGL committee, I have chance to participate the DAAD (Deutscher
Akademischer Austauschdienst) granted teaching assistantships (STIBET) and to share
my bioinformatics skills with other PhD students in life science. These 4 years are my
most valuable experience, so I tried my best to learn and to contribute my effort.
I would like to thank my Prof. Baumgart-Vogt very much for allowing me to
take this important step in my scientific career as a part of her team. I am thankful for
her help, her patience in all our discussions and for her advice during those past 4 years,
especially during my thesis correction period. She spent huge time and patience on my
English writing and experimental photos, gave me plenty of sugguestions to improve its
quality, and checked each point in detail to make sure all the information is correct. She
is a very responsible, reliable and sincere “Doktormutter”!
I am also thankful to Dr. Colasante. She has not only helped me a lot in my
experiments, such as the RNA isolation and precipitation, Northern blot “sandwiches”
set, washing buffer recipes, the principle of expression primer design and so on, but also
spent lots of time to guide and correct my thesis as well as the last proofreading.
Heartfelt thanks to Dr. Barbara Ahlemeyer for helping me to take photos from laser
scan confocal microscope. Thank sincerely my friend Francisca Alagboso for her great
help on the English correction of my whole thesis.
I am grateful for the friendly atmosphere in our team and lab and want to thank
my colleagues and technicians for the nice moments we could share in and besides
work. They are all nice and friendly and always the first one whom I share my joy and
tear with. Thanks to them, whenever I had trouble in my work or life, I could always get
suggestions and encouragement; thanks to them, I didn’t feel lonely in every birthday
Page | 159
and Christmas and lunch time; thanks to them, my album is full of smiling eyes and
sunny faces. Special thanks go to Frau Andrea Toxtor, who not only was a trusting
partner for my work, but also a great help for improving my German language. And
thank the technicians to help me in the cell culture and immunofluorescence, who also
lent German books to me for reading.
I also want to thank the Chinese government, because it would have been
impossible for me to complete my PhD studies in Germany without their scholarship.
Further I especially want to thank my mother, who always supported and
encouraged me to follow my interest and studies with her greatest love. I am also
grateful for all the support and patience I received from my family and friends,
especially my boyfriend who always encouraged me whenever needed and gave me
strength for the final steps in getting PhD.
Now it is almost the end of PhD study, however, it is also a new start to face
further chance and challenge. I believe I can be more brave and prepared to welcome
future. Because I know the concern and support is always surrounding closely.
Page | 160
14 Curriculum vitae
The curriculum vitae was removed from the
electronic version of the paper.
Page | 161
15 Reference
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A-kinase anchoring proteins AKAP1, -4, -10 and

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